Anal. Chem. 2008, 80, 9343–9348
Overcoming Matrix Effects in Liquid Chromatography-Mass Spectrometry Achille Cappiello,* Giorgio Famiglini, Pierangela Palma, Elisabetta Pierini, Veronica Termopoli, and Helga Trufelli University of Urbino “Carlo Bo”, Istituto di Scienze Chimiche “F. Bruner” 61029 Urbino, Italy A major limitation in quantitative analysis with electrospray ionization mass spectrometry (ESI-MS) is represented by the so-called matrix effects in which the matrix coextracted with the analytes can alter the signal response, causing either suppression or enhancement, resulting in poor analytical accuracy, linearity, and reproducibility. In the direct electron ionization liquid chromatography-mass spectrometry (direct-EI LC-MS) interface the ionization process is based on electron impact ionization, and it occurs in the gas phase and is not influenced by coeluted matrix compounds. In this work we quantitatively evaluated matrix effects on enriched environmental and biological samples, with different extraction procedures, using ESI and direct-EI LC-MS. As expected, the samples analyzed with direct-EI were not affected by matrix composition, whereas with ESI we observed either signal suppression or enhancement, depending on the sample nature. The combination of liquid chromatography and mass spectrometry into a single, well-integrated system (LC-MS) has revolutionized the approach to the quantitative determination of organic compounds in complex samples. Progress in life science research has been accomplished with the use of LC-MS, together with new applications of environmental, food safety, and homeland security interest.1-4 LC-MS systems using an electrospray ion source coupled with a tandem mass analyzer (LC-ESI-MS/MS) have gained a great popularity because of their effectiveness for the quantitative determination at trace level of medium- and highpolarity analytes. The high selectivity and sensitivity of modern MS/MS systems have led to a growing trend of high-throughput analyses that entail little or no sample preparation and a minimal chromatographic retention. Thanks to these important aspects, LC-ESI-MS/MS has become a well-established standard analytical technique in the pharmaceutical industry, clinical research, forensic analysis, environmental science, and in many other areas in which trace amounts of analytes in complex mixtures must be detected and characterized.2,3,5-7 However, in the past few years the common perception that the high selectivity of LC-MS/MS * Corresponding author. E-mail:
[email protected]. (1) Maurer, H. H. Anal. Bioanal. Chem. 2005, 381, 110–118. (2) Reemtsma, T. J. Chromatogr., A 2003, 1000, 477–501. (3) Prakash, C.; Shaffer, L.; Nedderman, A. Mass Spectrom. Rev. 2007, 26, 340–369. (4) Sforza, S.; Dall’Asta, C.; Marchelli, R. Mass Spectrom. Rev. 2006, 25, 54– 76. (5) Hopfgartner, G.; Bourgogne, E. Mass Spectrom. Rev. 2003, 22, 195–214. 10.1021/ac8018312 CCC: $40.75 2008 American Chemical Society Published on Web 10/30/2008
guarantees an effective elimination of the interferences from endogenous impurities has been challenged. Many authors report that the presence of coextracted matrix can severely affect the quantification procedures based on ESI and atmospheric pressure chemical ionization (APCI) LC-MS methods.8-12 This phenomenon is called matrix effects (ME), and it is considered to be either an unexpected suppression or enhancement of the analytes response induced by the coeluting matrix. It can heavily affect reproducibility, linearity, and accuracy of the method leading to erroneous quantitation.8,11 As a matter of fact, ME are becoming a major threat in the successful application of LC-ESI-MS, reducing the typical advantages of mass spectrometric detection in terms of selectivity and specificity, which is reminiscent of protocols used for less sophisticated detectors, such as ultraviolet or fluorescence. The exact mechanism of ME is still unknown. It is assumed that the coeluted matrix can influence signal intensity in a possible competition for the available charges and for the access to the droplet surface for gas-phase emission.13-16 Postcolumn infusion and postextraction addition are the two main strategies proposed in literature to asses ME. In the first one a pump is used to deliver a constant flow of analyte into the LC eluent at a point after the chromatographic column and before the mass spectrometer.17 A sample extract free of the analyte is then injected under the required chromatographic conditions, and the response from the infused analyte is recorded. Any endogenous matrix component that elutes from the column and induces ME can be seen as a suppression or enhancement of the infused analyte signal. In the postextraction addition a sample extract with the analyte of interest added after the extraction (matrix-matched (6) Alder, L.; Greulich, K.; Kempe, G.; Vieth, B. Mass Spectrom. Rev. 2006, 25, 838–865. (7) Wood, M.; Laloup, M.; Samyn, N.; del Mar Ramirez Feranandez, M.; de Bruijn, E. A.; Maes, R. A. A.; De Boeck, G. J. Chromatogr., A 2006, 1130, 3–15. (8) Matuszewski, B. K.; Constanzer, M. L.; Chevez-Eng, C. M. Anal. Chem. 2003, 75, 3019–3030. (9) Mei, H.; Hisieh, Y.; Nardo, C.; Xu, X.; Wang, S.; Ng, K.; Korrfmacher, W. A. Rapid Commun. Mass Spectrom. 2003, 17 (1), 97–103. (10) Antignac, J. P.; de Wasch, K.; Monteau, F.; De Brabander, H.; Andre, F. Anal. Chim. Acta 2005, 529, 129–136. (11) Taylor, P. J. Clin. Chem. 2005, 38, 328–334. (12) Niessen, W. M. A.; Manini, P.; Andreoli, R. Mass Spectrom. Rev. 2006, 25, 881–899. (13) Tang, L.; Kebarle, P. Anal. Chem. 1993, 65, 3654–3666. (14) Cech, N. B.; Henke, C. G. Anal. Chem. 2000, 72, 2717–2723. (15) King, R.; Bonfiglio, R.; Fernandez-Metzler, C.; Miller-Stein, C.; Olah, T. J. Am. Soc. Mass Spectrom. 2000, 11, 942–950. (16) Zhou, S.; Cook, K. D. J. Am. Soc. Mass Spectrom. 2001, 12, 206–214. (17) Bonfiglio, R.; King, R. C.; Olah, T. V.; Merkle, K. Rapid Commun. Mass Spectrom. 1999, 13, 1175–1185.
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standard) is compared with a pure solution containing the same amount of the target analyte.18 A difference in response between the matrix-matched standard and the pure solution indicates suppression or enhancement. Matuszewski et al. proposed several protocols to asses ME through the postextraction addition.8 These authors were also the first ones to introduce the terms “relative” and “absolute” ME. The “absolute ME” represents the difference in response between the standard and the postextraction spiked sample. The “relative ME” indicates the difference in response between various lots of postextraction spiked samples. The absolute ME affects the accuracy of the method, whereas the relative ME affects both accuracy and precision of the method. The validity of quantitative data obtained with an LC-MS method should be carefully verified by assessing absolute and relative ME. Several operational strategies have been suggested to minimize the interferences of coeluting matrix compounds. They include extensive cleanup procedures prior to LC-MS/MS analysis and more efficient chromatographic separations.10-12,19-23These strategies could help to reduce the introduction of matrix components into the analytical system, but they are laborious, time-consuming, and can lead to analyte loss. Another strategy involves the use of isotope-labeled internal standards (IS).10,12,20,24-26 In case of ME the ionization of the target analyte and the IS will be suppressed or enhanced at the same extent. However, the ratio of the two signals will not be affected and a correct quantification can be achieved. Nevertheless, it is worthwhile to notice that isotopelabeled standard are expensive and not always available. ME has been deeply investigated in the quantitation of drugs and their metabolites in plasma and other biological fluids;8,11,30-32 however, a great deal of attention has been driven also to environmental applications.12,21,33,34 An efficient LC-MS interface based on electron ionization (direct-EI) has been presented for the analysis of small-medium molecular weight molecules.27-29 In two environmental applications we already observed that direct-EI is not affected by ME.35,36 In this work we demonstrate it quantitatively on two different (18) Buhrman, D.; Price, P. I.; Rudewicz, P. J. J. Am. Soc. Mass Spectrom. 1996, 7, 1099–1105. (19) Pascoe, R.; Foley, J. P.; Gusev, A. I. Anal. Chem. 2001, 73, 6014–6023. (20) Avery, M. Rapid Commun. Mass Spectrom. 2003, 17, 197–201. (21) Benijts, T.; Dams, R.; Lambert, W.; De Leenher, A. J. Chromatogr., A 2004, 1029, 153–159. (22) Van De Steene, J. C.; Mortier, K. A.; Lambert, W. E. J. Chromatogr., A 2006, 1123, 71–81. (23) Kang, J.; Hick, L. A.; Price, W. E. Rapid Commun. Mass Spectrom. 2007, 21, 4065–4072. (24) Zrostlikova´, K. H.; Poutska, J.; Begany, P. J. Chromatogr., A 2002, 973, 13–26. (25) Toussaint, B.; Bordin, G.; Janosi, A.; Rodriguez, A. M. J. Chromatogr., A 2002, 976, 195–206. (26) Amnesley, T. M. Clin. Chem. 2003, 49, 1041–1044. (27) Cappiello, A.; Famiglini, G.; Mangani, F.; Palma, P. J. Am. Soc. Mass Spectrom. 2002, 13, 265–273. (28) Cappiello, A.; Famiglini, G.; Palma, P. Anal. Chem. 2002, 75, 497A–503A. (29) Cappiello, A.; Famiglini, G.; Palma, P.; Pierini, E.; Trufelli, H. Anal. Chem. 2007, 79, 5364–5372. (30) Mei, H.; Hsleh, Y. H.; Nardo, C.; Xlaoyng, X.; Wang, S.; Ng, K.; Korfmacher, W. Rapid Commun. Mass Spectrom. 2003, 17, 97–109. (31) Dams, R.; Huestis, M. A.; Lambert, W. E.; Murphy, C. M. J. Am. Soc. Mass Spectrom. 2003, 14, 1290–1294. (32) Liang, H. R.; Foltz, R. C.; Meng, M.; Bennet, P. Rapid Commun. Mass Spectrom. 2003, 17, 2815–2821. (33) Schu ¨ sener, M. P.; Bester, K. Rapid Commun. Mass Spectrom. 2005, 19, 3269–3278. (34) Zhao, X.; Metcalfe, C. D. Anal. Chem. 2008, 80, 2010–2017.
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complex matrixes: human plasma and river water. Two drugs (ibuprofen and phenacetin) and four pesticides (methomyl, aldicarb, atrazine, and propazine) were chosen as target compounds and analyzed with direct-EI and ESI using different extraction procedures. EXPERIMENTAL SECTION Analysis of Plasma Samples. Standard Solutions. All solvents were LC grade from VWR International (Milan, Italy). Reagent grade water was obtained from a Milli-Q water purification system (Millipore, Bedford, MA). Phenacetin and ibuprofen were purchased from Sigma-Aldrich (Milan, Italy). Stock solutions at 10 mg/L were prepared dissolving each analyte in 100% ethanol. A working solution in ethanol/water (10/90, v/v) containing ibuprofen at 1 mg/L was used for spiking the dried extracts to be analyzed by both LC-direct-EI-MS and LC-ESI-MS. Two different concentrations were used for phenacetin spiked solutions: (1) 0.1 mg/L standard solution in ethanol/water (10/90, v/v) with 0.1% formic acid was used for spiking the extracts analyzed by LC-ESIMS/MS; (2) extracts to be analyzed by LC-direct-EI-MS were spiked with a working solution at 1 mg/L (ethanol/water, 10/90, v/v). Sample Preparation. Human plasma samples underwent to liquid-liquid (LL) extraction and solid-phase extraction (SPE). LL extraction was carried out following the protocol described by Bonfiglio et al.17 A volume of 3 mL of methyl-tert-butyl ether (MTBE) was added to 500 µL of human plasma, and the mixture was centrifuged at 3000 rpm for 10 min. The supernatant was filtered with poly(tetrafluoroethylene) (PTFE) filters (pore size 0.22 µm) and transferred into a clean Teflon test tube. The extract was evaporated to dryness under nitrogen and then reconstituted in 250 µL of the analyte spiking solution. Two different SPE procedures were followed.17,37 The extraction procedure for the analysis of phenacetin was carried out using Oasis HLB (100 mg) cartridges from Waters Corporation (Milford, MA).17 The cartridges were preconditioned with 2 mL of methanol followed by 2 mL of water. A volume of 1 mL of blank plasma sample was centrifuged at 2000 rpm for 5 min, and the supernatant separated. The sample was then forced through the cartridge at a flow rate of about 1 mL/min. The cartridge was rinsed with 0.5 mL of water and 1 mL of methanol/water (5/95, v/v). Finally, the sample was eluted with 2 mL of methanol and the eluate was evaporated to dryness under a stream of nitrogen at room temperature. The residue was dissolved in 1 mL of the phenacetin spiking solution. The SPE of ibuprofen was carried out referring the procedure of Vinci et al.37 using Agilent AccuBondII SPE ODSC18 200 mg (Agilent Technologies, Santa Clara, CA). A volume of 5 mL of plasma was centrifuged at 2000 rpm for 5 min, and the supernatant separated. Then, 500 µL of 1 mol/L HCl was added, to adjust the pH at about 3.0, and the solution was allowed to stand for 10 min at room temperature. This step allows both the denaturation of plasma proteins and the hydrolysis of bound drug residues. The sample was then added of 25 mL of ascorbic acid (35) Cappiello, A.; Famiglini, G.; Palma, P.; Pierini, E.; Maggi, C.; Manfra, L.; Mannozzi, M. Chemosphere 2007, 69, 554–560. (36) Famiglini, P.; Palma, P.; Pierini, P.; Cappiello, A. Anal. Chem. 2008, 80, 3445–3449. (37) Vinci, F.; Fabbrocino, S.; Fiori, M.; Serpe, L.; Gallo, P. Rapid Commun. Mass Spectrom. 2006, 20, 3412–3420.
buffer 0.010 mol/L at pH 3.0 (AA buffer) and loaded at a flow rate of about 1 mL/min onto a C18 1 g SPE cartridge, previously rinsed and equilibrated with 3 mL of methanol and 5 mL of AA buffer. After loading, the SPE cartridge was washed with 3 mL of AA buffer and 3 mL of Milli-Q water and then dried under vacuum for 10 min. Finally, the SPE cartridge was eluted with 3 mL of n-hexane/diethyl ether (50/50, v/v), and the eluate was evaporated to dryness under a stream of nitrogen at room temperature. The residue was dissolved in 1 mL of the ibuprofen spiking solution. LC-ESI-MS/MS Conditions. Separations were performed, using a SpectraSystem P2000 binary gradient pump (Thermo Fischer Scientific, San Jose, CA), at a flow rate of 300 µL/min in a 250 mm × 2.1 mm Supelcosil LC18 column packed with a 5 µm phase (Supelco, Bellefonte, PA). The standard and extracts spiked with ibuprofen were analyzed using a gradient elution program with water (solvent A) and acetonitrile (solvent B). Experiments regarding the ME evaluation on phenacetin were carried out using water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B) in gradient elution from 100% to 0% A in 20 min. The injection volume was 5 µL. The eluted drugs were introduced into an ESI-ion trap mass spectrometer (LCQDuo, Thermo Fischer Scientific, San Jose, CA). Data acquisition was carried out in selected reaction monitoring (SRM) using the following settings. (1) Detection of ibuprofen: negative ion mode; spray voltage 4.5 kV; sheath gas 60 psi; auxiliary gas pressure 20 psi; heated capillary voltage and temperature -4 V and 225 °C, respectively. During SRM experiments (transition from m/z 205 to m/z 159), ions were accumulated into the trap for 800 ms, while three microscans were summed up to provide the final signal. Collision energy was set at 25%. (2) Detection of phenacetin: positive ion mode; spray voltage 4.5 kV; sheath gas pressure 60 psi; auxiliary gas pressure 20 psi; heated capillary voltage and temperature 10 V and 225 °C, respectively. During SRM experiments (transition from m/z 180 to m/z 138), ions were accumulated in the trap for 800 ms, while three microscans were summed up to provide the final signal. Collision energy was set at 30%. Experiments for method performance evaluation on standard solution were carried out in flow injection mode (FIA) mode at a flow rate of 300 µL/min, injecting a volume of 5 µL. The mobile phase was composed of water/acetonitrile (50/50, v/v) for the analysis of ibuprofen and water/acetonitrile (50/50, v/v) added with 0.1% formic acid for phenacetin. LC-Direct-EI-MS Conditions. Liquid chromatography was performed on an Agilent 1100 series nanoLC system (Agilent Technologies, Santa Clara, CA) using an Agilent C18 Zorbax-SB column (150 mm × 75 µm i.d., 3.5 µm particle size). Injection volume was 60 nL, and a 20 min gradient from 100% water to 100% acetonitrile was delivered at a flow rate of 300 nL/min. The directEI interface was mounted on an Agilent 5975B Inert MSD single quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA). A detailed description of the direct-EI interface is reported elsewhere.29 Data acquisition during the chromatographic separations was carried out in selected ion monitoring (ibuprofen. m/z 119, 163, 206; phenacetin. m/z 108, 137, 179) using the following
settings: ion source temperature 280 °C; quadrupole temperature 150 °C; dwell time 500 ms; cycles/s 0.66. Experiments for the method performance evaluation were carried out in FIA mode at a flow rate of 300 nL/min, injecting a volume of 60 nL. For both drugs mobile phase was composed of water/acetonitrile (50/50, v/v). During the postcolumn infusion experiments, mobile phase was delivered into the direct-EI interface at a flow rate of 300 nL/min, and the analyte was infused through a zero dead volume tee using a Kontron HPLC pump 422 (Kontron Instruments, Milan, Italy). The blank plasma extracts were injected onto the Zorbax-SB column using the chromatographic conditions reported previously. Effluent from the column combined with the infused analyte and entered into the direct-EI interface. Analysis of Water Samples. Chemicals and Reagents. Methanol, MTBE, 2-propanol, and HPLC grade acetonitrile were purchased from VWR (Milan, Italy). Atrazine, aldicarb, methomyl, and propazine were purchased from Sigma-Aldrich Chemicals (Milan, Italy). The Suwannee river fulvic acid was purchased from the International Humic Substances Society (IHSS, http://ihss. gatech.edu/). Stock solutions of each pesticide at a concentration of 10 mg/L were prepared in acetonitrile. The artificial matrix was prepared diluting the Suwannee river fulvic acid to a final concentration of 100 mg/L. This balanced composition well represents the average polluted river sample in predictable and controlled laboratory conditions. Each pesticide was dissolved in the artificial matrix to a final concentration of 10 mg/L. River Water SPE Procedure. SPE was carried out following the protocols of Benijts et al.21 An OASIS HLB (6 mL, 200 mg) (Waters, Milford, MA) cartridge was installed on a Visiprep SPE vacuum manifold (Supelco, Bellefonte, PA). The cartridges were previously preconditioned with 6 mL of 2-propanol/MTBE (10/ 90, v/v), followed by 6 mL of methanol and 6 mL of water. The aqueous samples (1000 mL) were loaded at a flow rate of 10 mL/ min. Then, the cartridge was rinsed with 3 mL of a mixture of water and methanol (70/30, v/v), 3 mL of water, and 3 mL of 2% ammonia/methanol (90/10 v/v, pH 11.5). The cartridge was dried under vacuum for 30 min, and the analytes were eluted with 6 mL of a 2-propanol/MTBE (10/90, v/v) mixture. Finally, the eluate was evaporated to dryness under a gentle stream of nitrogen. The dry residue was dissolved in 500 µL of a water/acetonitrile (95/ 5, v/v) standard solution containing the target pesticides at the following concentrations: methomyl and aldicarb at 1 mg/L and atrazine and propazine at 0.1 mg/L. LC-ESI-MS/MS Analysis. The ME evaluation on the artificial matrix was carried out injecting standards and sample in FIA mode. The mobile phase used to drive the samples into the mass spectrometer was composed of water/acetonitrile (50/50, v/v). The flow rate and the injection volume were of 300 µL/min and 5 µL, respectively. The same conditions were used for the experiments regarding method validation. LC separations were achieved on a Waters NovaPack C18 column (150 mm × 2.1 mm, 3.5 µm) (Waters, Milford, MA). Eluent flow rate was set at 250 µL/min with a mobile phase composed of water (solvent A) and acetonitrile (solvent B) in gradient elution from 90% to 10% A in 40 min. Injection volume was 5 µL. The flow rate was sent to an ion trap mass spectrometer with an ESI source (LCQDuo, Thermo Fischer Scientific, San Jose, CA). During the LC-ESI-MS/MS Analytical Chemistry, Vol. 80, No. 23, December 1, 2008
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Table 1. LC-ESI-MS/MS Calibration Data compd
LOD
LOQ
range of linearity
linear regression equation
aldicarb atrazine ibuprofen methomyl phenacetin propazine
50 pg 10 pg 2.5 ng 50 pg 50 pg 25 pg
167 pg 33 pg 9.3 ng 167 pg 167 pg 83 pg
50 pg to 5 ng 10 pg to 50 ng 2.5 ng to 250 ng 50 pg to 50 ng 50 pg to 50 ng 5 pg to 50 ng
Y Y Y Y Y Y
) 12145X + 74719 ) 22669X - 77778 ) 103071X + 656153 ) 7313.5X - 248661 ) (4 × 109)X - 1 × 109 )26947X + 810072
R2
intraday precision (% RSD)
interday precision (% RSD)
0.9962 0.9998 0.9835 0.9993 0.9952 0.9986
5 8 9 7 8 5
10 10 15 11 15 12
R2
intraday precision (% RSD)
interday precision (% RSD)
0.9999 0.9999 0.9990 0.9994 0.9997 0.9984
6 5 6 6 5 4
8 6 8 8 10 6
Table 2. LC-Direct-EI-MS Calibration Data compd
LOD
LOQ
range of linearity
aldicarb atrazine ibuprofen methomyl phenacetin propazine
60 pg 10 pg 30 pg 60 pg 30 pg 20 pg
200 pg 33 pg 100 pg 200 pg 100 pg 67 pg
60 pg to 600 ng 10 pg to 10 ng 30 pg to 30 ng 60 pg to 600 ng 30 pg to 30 ng 20 pg to 60 ng
linear regression equation Y Y Y Y Y Y
) ) ) ) ) )
2772.2X - 1660.4 1274.4X + 1605.9 14930X - 63353.0 32336.3X - 8546.1 23489.0X + 74638 1591.7X - 7594.8
experiments, mass spectra were acquired in positive ion mode. The following experimental operating parameters were used: capillary temperature 225 °C; spray voltage 4.5 kV; sheath gas 60 psi; auxiliary gas 20 psi. Data acquisition during the chromatographic separations was carried out in SRM mode following these experimental conditions: (1) from 0 to 10 min monitoring of methomyl (transition from m/z 185 to 128); (2) from 10 to 16 min monitoring of aldicarb (transition from m/z 213 to 116); (3) from 16 to 20 min monitoring of atrazine (transition from m/z 216 to 176); (4) from 20 min monitoring of propazine (transition from m/z 231 to 188). LC-Direct-EI-MS. The liquid chromatography apparatus was the same as that used for plasma samples. Experiments in FIA mode were carried out at a flow rate of 300 nL/min. Mobile phase was composed of water/acetonitrile (50/50, v/v), and the injection volume was 60 nL. For more flexibility the autoinjector was replaced with a manual injector from Valco, equipped with a 500 nL internal loop (Valco, Houston, TX). For on-column experiments the mobile phase was composed of water (A) and acetonitrile (B) in gradient elution from 100% A to 100% B in 40 min. Scan time for full spectrum acquisition was adjusted in order to obtain a mean of 15 acquisition samples of each HPLC peak (1.5-1.8 scan/s). Data acquisition during the chromatographic separations was carried out in selected ion monitoring (SIM) using the following conditions: (1) from 0 to 15.5 min monitoring of methomyl (m/z 105-88); (2) from 15.5 to 20.5 min monitoring of aldicarb (m/z 115, 100, 89); (3) from 20.5 to 24.0 min monitoring of atrazine (m/z 215, 200, 173); (4) from 24.0 min monitoring of propazine (m/z 229, 214). Dwell times were chosen on the basis of the number of ions present in each ion program in order to collect a minimum of 20 acquisition samples for each HPLC peak (0.7-1 cycles/s). RESULTS AND DISCUSSION Prior to ME assessment, method validation was performed, including sensitivity, linearity, and precision. The experiments on neat standard solutions were carried out in FIA mode using the conditions reported in the Experimental Section. Sensitivity was expressed by the instrumental limit of detection (LOD) and limit of quantitation (LOQ) for each target compound. LOD and LOQ 9346
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were defined as the lowest analyte amount with a signal-to-noise ratio (S/N) of 3 and 10, respectively. LODs and LOQs are reported in Tables 1 and 2, and as can be inferred by the data, the sensitivity of the two analytical systems is comparable for most compounds. To asses linearity five points calibration curves were calculated over the linear range of the instrument response. Linear regression plots were calculated using peak area versus absolute injected amount; five replicates were analyzed for each concentration. Linearity was expressed as the squared correlation coefficient (R2). As reported in Tables 1 and 2, values higher than 0.9950 were obtained for most compounds. Precision was calculated as intraday and interday repeatability and expressed as the relative standard deviation (% RSD) at a concentration level used for the spiking procedure of the extracts (see the Experimental Section). Intraday repeatability was assessed analyzing in FIA five replicates in one day. Interday repeatability was evaluated on column, injecting three replicates on five different days (n ) 15). Intraday and interday repeatability of the method provided % RSD values e15% for all compounds, showing a satisfactory precision at the selected concentration levels. Ibuprofen and phenacetin were used as model compounds for the evaluation of ME in human plasma. Prior to LC-MS analysis, plasma samples were cleaned up following two different procedures: liquid-liquid extraction and solid-phase extraction. ME was evaluated using the postextraction addition method, which is based on the comparison of the signal of an analyte in a pure solution and the matrix-matched standard.8,18 As reported in the introduction, this procedure prevents any recovery miscalculation and focuses the response only to ME. As proposed by Matuszewski et al.,8 the absolute ME was calculated in percent as the ratio between the average peak area of the sample spiked after extraction (n ) 3) and the average peak area of the neat standard solution (n ) 3), multiplied by 100. In this context, a value >100% indicates ionization enhancement, whereas a value
