Anal. Chem. 2006, 78, 1331-1336
Method for Internal Standard Introduction for Quantitative Analysis Using On-Line Solid-Phase Extraction LC-MS/MS Yazen Alnouti, Ming Li, Olga Kavetskaia, Honggang Bi, Cornelis E. C. A. Hop, and Arkady I. Gusev*
Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Groton, Connecticut 06340
A novel approach for on-line introduction of internal standard (IS) for quantitative analysis using LC-MS/MS has been developed. In this approach, analyte and IS are introduced into the sample injection loop in different steps. Analyte is introduced into the injection loop using a conventional autosampler (injector) needle pickup from a sample vial. IS is introduced into the sample injection loop on-line from a microreservoir containing the IS solution using the autosampler. As a result, both analyte and IS are contained in the sample loop prior to the injection into the column. Methodology allowed to reliably introduce IS and demonstrated injection accuracy and precision comparable to those obtained using off-line IS introduction (i.e., IS and analyte are premixed before injection) while maintaining chromatographic parameters (i.e., analyte and IS elution time and peak width). This new technique was applied for direct analysis of model compounds in rat plasma using on-line solid-phase extraction (SPE) LC-MS/MS quantification. In combination with on-line SPE, IS serves as a surrogate IS and compensates for signal variations attributed to sample preparation and instrumentation factors including signal suppression. The assays yielded accuracy (85-119%), precision (2-16%), and analyte recovery comparable to those obtained using off-line IS introduction. Furthermore, on-line IS introduction allows for nonvolumetric sample (plasma) collection and direct analysis without the need of measuring and aliquoting a fixed sample volume prior to the on-line SPE LC-MS/MS analysis. Therefore, this methodology enables direct sample (plasma) analysis without any sample manipulation and preparation. Quantitative analysis using liquid chromatography-mass spectrometry (LC-MS/MS) requires external or internal standard (IS) to provide quantitative reference calibration points.1-3 Although LC-MS/MS quantification using external standard has been demonstrated,1,2,4,5 IS applications are usually considered more * To whom all correspondence should be addressed. Phone (860) 715-3009. E-mail:
[email protected]. (1) Niessen, W. M. A. Rev. Anal. Chem. 2000, 19, 289-301. (2) Plumb, R. S.; Dear, G. J.; Mallet, D. N.; Higton, D. M.; Pleasance, S.; Biddlecombe, R. A. Xenobiotica 2001, 31, 599-617. (3) Zimmer, D. Chromatogr. Suppl. 2003, 57, S325-S332. (4) Perico, A.; Balglioni, S.; Bavazzano, P. Rapid Commun. Mass Spectrom. 2004, 18, 865-1868. 10.1021/ac051806q CCC: $33.50 Published on Web 01/19/2006
© 2006 American Chemical Society
versatile and eliminate a number of problems encountered in quantitative analysis including variability in sample preparation (e.g., dilution, evaporation, reconstitution, and extraction recovery) and instrumental variability (e.g., matrix effect and injection volume).6,7 To be effective, the internal standard must have physicochemical properties similar to that of the analyte. For this reason, isotopically labeled compounds are considered to be the “ideal” IS.3 However, occasionally isotopically labeled IS may also lead to analyte signal suppression and enhancement.8,9 In addition, isotopically labeled IS may not be readily available for some analytes. Therefore utilization of analyte structural analogues as IS has become routine practice.9 There are two common methods by which the IS can be introduced into the sample.7 The volumetric introduction method involves the addition of IS after sample extraction and cleanup but prior to analysis to address instrumental errors (e.g., injection volumes, signal/ionization suppression, and enhancement). The surrogate introduction method involves addition of the IS prior to any sample extraction procedure. Surrogate IS compensates for signal variability attributed to sample preparation (e.g., extraction, purification, and cleanup) as well as addresses instrumentation deviations. Both surrogate and volumetric introduction methods usually require a separate off-line step for manual or automated addition of IS to the sample. Alternatively, IS can be introduced on-line prior to analysis. On-line method of IS introduction using continuous postcolumn or precolumn infusion has been demonstrated to specifically address instrument instability, signal drift issues, and compensate for signal suppression and enhancement in LCMS/MS analysis.10,11 Continuous infusion of IS allowed the application of a single IS for the quantification of multicomponent mixtures with high accuracy and precision.10 On-line addition of the IS for flow injection analysis was also demonstrated for ICPMS (5) Sabatini, L.; Barbieri, A.; Tosi, M.; Roda, A.; Violante, F. S. Rapid Commun. Mass Spectom. 2005, 19, 147-152. (6) Rosing, H.; Man, W. Y.; Doyle, E.; Bult, A.; Beijnen, J. H. J. Liq. Chromatogr. Relat. Technol. 2000, 23, 329-354. (7) Boyd, R. K. Rapid Commun. Mass Spectrom. 1993, 7, 257-271. (8) Liang, H. R.; Foltz, R. L.; Meng, M.; Bennett, P. Rapid Commun. Mass Spectrom. 2003, 17, 2815-2821. (9) Stokvis, E.; Rosin, H.; Beijnen, J. H. Rapid Commun. Mass Spectrom. 2005, 19, 401-407. (10) Choi, B. K.; Gusev, A. I.; Hercules, D. M. Anal. Chem. 1999, 71, 41074110. (11) Choi, B. K.; Hercules, D. M.; Gusev, A. I. Fresenius J. Anal. Chem. 2001, 369, 370-377.
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Table 1. Sample Extraction Process As Performed by the Symbiosis System solvent equilibration 1 equilibration 2 loadingb washing elution
ammonium acetatea acetonitrile ammonium acetatea ammonium acetatea acetonitrile
flow rate volume duration (mL/min) (mL) (min) 5 5 2 5 0.14
1 1 0.5 1 0.2
0.3 0.3 0.35 0.3 1.5
a 5 mM ammonium acetate buffer (pH 7). b In this step, the sample is transferred from the injector loop to the SPE cartridge.
Figure 1. Chemical structures of propranolol, ketoconazole (IS), diclofenac, and ibuprofen (IS).
analysis of various metals in drinking water using the method of standard addition. Sequential injections of internal standard solutions are introduced through a separate valve and combined with the sample solution on-line entering the MS source.12,13 A similar approach was also used during ICR-MS mass calibration. Internal standard and analyte can be sequentially introduced via a dual ESI source to achieve high mass accuracy.14 An approach of “deferred standards” for on-line introduction of IS has also been developed for HPLC-UV analysis. Double injections of the sample and the internal standard are performed via one injection valve with a delay time to ensure complete separation of the internal standard peak and the sample peaks. The deferred standard allowed for continuous monitoring of system chromatographic performance.15 This paper reports a new method of on-line IS introduction specifically for LC-MS analysis. In this approach, analyte and IS are separately introduced into the sample injection loop. First, analyte is introduced into the injection loop using a conventional autosampler (injector) needle pickup from a sample vial. Second, IS is introduced into the sample injection loop on-line from a microreservoir containing the IS solution using the autosampler. As a result, both analyte and IS are contained in the sample loop prior to the injection into the column. In combination with online solid phase extraction (SPE), IS serves as a surrogate IS and compensates for signal variations attributed to both sample preparation including extraction recovery and instrumentation factors including signal suppression. To demonstrate the utility of the new approach and provide comparison with off-line addition of IS, analytical methods to quantify model compounds, i.e., propranolol and diclofenac with IS, ketoconazole, and ibuprofen, respectively, in rat plasma using LC-MS/MS assays were fully developed and validated. EXPERIMENTAL SECTION Chemicals and Materials. Propranolol, diclofenac, ibuprofen, and ketoconazole were purchased from Sigma (St.Louis, MO). Figure 1 shows the chemical structures of propranolol and (12) Pinto, M. S.; Silva, P.; Masini, J. C. Anal. Chim. Acta 2002, 466, 345-352. (13) Wiederin, D. R.; Smyczek, R. E.; Houk, R. S. Anal. Chem. 1991, 63, 62631. (14) Hannis, J. C.; Muddiman, D. C. Am. Soc. Mass Spectrom. 2000, 11, 876883. (15) Clarot, I.; Dreyfuss, M. F.; Domelier, R. Battu, S.; Cardot, P. J. J. Chromatogr., A 2002, 958, 79-88.
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diclofenac as well as their IS, ketoconazole and ibuprofen, respectively. These compounds have diverse physicochemical properties that represent a broad spectrum of typical small molecules encountered in pharmaceutical analysis. Rat plasma was obtained from Biochemed Pharmacologicals (Winchester, VA). Standard laboratory safety procedures handling biological samples were followed during this project. HPLC grade methanol, acetonitrile, and water were obtained from J.T. Baker (Philipsburg, NJ). Ammonium acetate, ammonium formate, formic acid, acetic acid, and ammonium hydroxide were purchased from J.T. Baker. C18 Luna column (2.1 × 50 mm, 5 µm) was purchased from Phenomenex (Torrance, CA). The C18 HD cartridges (2 × 10 mm) were obtained from Spark Holland. Chromatographic Conditions. The Symbiosis system (Spark Holland) was used for on-line SPE. C18 Luna (2.1 × 50 mm, 5 µm) analytical column was used in this study. The injection volume was 10 µL. The flow rate was set at 0.8 mL/min. The mobile phase consisted of solvent A (5 mM ammonium acetate buffer, pH 7) and solvent B (acetonitrile). The LC gradient flow was programmed at 5% B to 1.5 min, increased to 65% in 15 s, maintained at 65% for 55 s, increased to 90% in 5 s, maintained at 90% for 50 s, and decreased to 5% in 5 s, where the column was equilibrated for 20 s. Total run time including equilibration was 3 min and 25 s. Mass Spectrometry Conditions. Mass Spectrometry detection was carried out using the ESI mode with an API 4000 (ABSciex, Concord, ON, Canada) triple quadruple system. Propranolol and ketoconazole were analyzed in the positive ionization mode, while the negative ionization mode was used for diclofenac and ibuprofen. MS parameters to obtain the highest signal of all analytes are provided in Supporting Information. Sample Extraction. The C18 HD (2 × 10 mm) was the SPE cartridge of choice because it yielded the highest recovery, retention, and satisfactory peak shape for both propranolol and diclofenac. Table 1 lists the steps of the sample extraction process as performed by the Symbiosis system. The on-line sample extraction procedure is discussed in detail elsewhere.16 Analyte recoveries were determined by normalizing the peak area of analytes in plasma samples to samples prepared in neat solution. Preparation of Standards and Calibration Curves with OffLine Addition of the IS. Stock solutions of propranolol, ketoconazole, diclofenac, and ibuprofen were prepared in ACN at 1 mg/mL concentrations. Analyte (propranolol and diclofenac) standard solutions were prepared from the stock solutions by serial dilution with 5% ACN in HPLC grade water.
Figure 2. (Steps 1 and 2) The syringe aspirates 75 µL of the IS solution from the stock bottle and then fills a special reservoir vial in the autosampler. (Step 3) The injector needle aspirates 37.5 µL from the IS reservoir vial while the injection valve is in the inject position. Therefore, none of the IS solution has entered the loop yet. However, the injection needle and the connection tubes up to the injection valve are filled with the IS solution. Total volume of the needle and the connection tube is 15 µL. (Step 4) The injection valve switches to the load position and the injector needle aspirates 10 µL of sample from the 96-well plate while the injection valve is in the inject position. The aspirated sample has displaced 10 µL of IS present in the connection tube into the loop. (Step 5) The injector needle aspirates 37.5 µL from the IS reservoir vial while the injection valve is in the load position. There is 5 µL of IS solution remaining in the connection tube from the previous step, 10 µL of sample and 22.5 µL of IS solution (37.5 µL aspirated - 15 µL connection tube content). Therefore, the loop contains 10 µL of sample sandwiched between 15-µL leading and 22.5-µL tailing IS plugs (total 37.5 µL of IS). (Step 6) The injection valve switches to the inject position, where the loaded loop is on-line with the LC flow.
Blank rat plasma (1 mL) was spiked with 10 µg/mL IS (ketoconazol and ibuprofen, for propranolol and diclofenac analytes, respectively) solution and the proper analyte standard solution, 50 µL each, to prepare the calibration curve and the quality control (QC) points. Calibration curves were constructed of the following concentrations: 1, 2, 10, 50, 100, 300, 900, and 1000 ng/mL. Five QC points of the concentrations 1, 3, 500, 800, and 1000 ng/mL were used to validate the calibration curves. The IS concentration, ketoconazole for the propranolol method and ibuprofen for the diclofenac method, was 500 ng/mL. Preparation of Standards and Calibration Curves with OnLine Addition of the IS. Sample preparation was identical to the off-line addition of IS except that no IS was added to the sample.
The IS concentration in each sample was targeted to be 500 ng/ mL. The injection volume was 10 µL. Therefore, the injected samples contained 5 ng of IS. The volume of the IS solution injected in the “on-line IS addition” mode is fixed in the Symbiosis system, which equals 37.5 µL. Therefore, a 133.3 ng/mL standard IS solution was prepared to contain 5 ng of IS in each 37.5-µL injection. The 133.3 ng/mL IS standard solutions were prepared in 5% ACN for ketoconazole (IS for propranolol) and ibuprofen (IS for diclofenac) and introduced by the austosampler. Mechanism of On-Line Addition of IS. Figure 2 illustrates the process of on-line addition of IS in the Symbiosis system, step by step. The internal standard solution is placed in a primary reservoir located outside the autosampler, which supplies a special Analytical Chemistry, Vol. 78, No. 4, February 15, 2006
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Figure 3. LC-MS chromatograms of propranolol (50 ng/mL) with on-line (A) and off-line (B) introduction of ketoconazole (IS) (500 ng/mL); and diclofenac (50 ng/mL) with on-line (C) and off-line (D) introduction of ibuprofen (IS) (500 ng/mL).
vial (microreservoir) built inside the autosampler. In the first two steps, the syringe pump is programmed to fill the microreservoir inside with IS solution (Figure 2, steps 1 and 2). The injection needle then aspirates half of the IS microreservoir content (37.5 µL) into the needle tubing (15 µL), filling it with IS solution. The rest of the aspirated IS will go to waste since the injection valve is on the inject position. None of the IS solution has entered the loop yet. But, the needle and the connection tube is now full with IS (Figure 2, step 3). The injection valve switches to load position, and the injector needle aspirates 10 µL of sample from the 96well plate, displacing 10 µL of IS that is present in the connection tube and pushing it into the loop. At this stage, the needle and the connection tube contains 5 µL of IS (15-10 µL) in addition to a 10-µL sample. No sample has entered the loop yet (Figure 2, step 4). In the next step, the second half of the IS microreservoir content (37.5 µL) is aspirated into the needle tubing, pushing its content into the injection loop. This will displace the 15-µL (5 µL of IS + 10 µL of sample) needle and connection tubing content into the loop. Also 22.5 µL of IS (37.5 µL aspirated -15 µL left in connection tubing) will enter the loop. As a result, the loop contains 10 µL of sample sandwiched between 15-µL leading IS plug and 22.5-µL tailing IS plug (37.5 µL of total IS) (Figure 2, step 5). Finally, the injection valve switches to the inject position, in which the loaded loop is on-line with the LC flow (Figure 2, step 6). Method Development and Validation. Method development, including SPE cartridge selection and extraction condition optimization, is an automatic process using the Symbiosis system, which was described somewhere else.16 Intraday and interday accuracy and precision as calculated from five QC points were (16) Alnouti, Y.; Srinivasan, K.; Waddell, D.; Bi, H.; Kavetskaia, O.; Gusev, A. I. J. Chromatogr., A 2005, 1080, 99-106.
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used to validate the calibration curves. Intraday precision was calculated as percent relative standard deviation (% RSD) of the analyte to internal standard peak areas obtained from replicates (n ) 5) of each QC point. Accuracy was calculated as the percent bias of the calculated concentration relative to the nominal concentration of each QC point. The process was repeated over 3 days using freshly prepared standards. Results from the 3 days were pooled to calculate interday accuracy and precision (n ) 15). RESULTS AND DISCUSSION Approach. This paper reports a novel method of on-line IS introduction specifically for LC-MS analysis. The autosampler can be used in two different ways to introduce IS on-line. Analyte and IS can sequentially be aspirated by injector needle from two different vials (i.e., sample vial and IS vial). In this approach, the needle will contain both analyte and IS and both can be injected into the sample loop at the same time. Although a viable approach, repetitive injections from the sample and IS vials can potentially lead to cross contamination of sample and IS vials. To avoid cross contamination, IS can alternatively be introduced into the injection loop directly from an individual reservoir connected to the injector port. This can be achieved utilizing one of the injection modes (“microliter-pickup” mode) provided by the Symbiosis autosamler, which is designed to minimize the volume of wasted sample during sample injection. In this injection mode, the amount of sample used in any injection equals exactly the sample injection volume with no extra sample used to deliver the sample to the injection loop through the dead volume of the injection needle and tubing. A transfer solvent compatible with the sample matrix is used to fill the injection needle and the tubing and carry the sample plug to the injection loop. In this report, we replaced the transfer solvent with internal standard solution and
Table 2. Validation Results Obtained for Propranolol and Diclofenac Off-Line Propranolol day 2 day 3 accuracy precision accuracy precision
day 1
QC (ng/mL)
accuracya
precisionb
1 3 75 500 1000
104.2 93.7 96.2 93.5 102.2
8.1 4.6 2.0 11.1 12.1
86.0 112.3 116.6 109.8 111.4
accuracya
precisionb
1 3 75 500 1000
110.4 108 108.8 87.5 90.5
5.0 2.7 2.7 3.1 3.8
100.9 109.5 105.0 86.6 104.9
9.9 4.4 5.0 2.2 7.6
On-Line Propranolol day 2 day 3 accuracy precision accuracy precision
day 1
QC (ng/mL)
3.2 3.4 2.0 3.0 2.5
105.1 104.0 98.2 89.1 85.0
on-line
QC (ng/mL)
accuracya
precisionb
1 3 75 500 1000
103.9 101.0 101.0 95.1 106.3
13.4 2.2 2.5 6.8 6.4
16.0 6.8 3.5 0.7 4.0
93.1 92.0 118.8 104.4 99.5
9.8 5.7 1.9 3.9 3.5
interdayc accuracy precision 97.0 105.9 105.9 97.5 104.9
11.8 8.5 8.4 12.0 7.3
interdayc accuracy precision 102.2 101.5 105.9 93.3 91.5
12.1 8.2 7.5 8.8 8.5
Diclofenac off-line accuracy precision 98.8 98.8 98.3 98.1 100.2
9.9 6.4 2.1 6.2 5.7
a Accuracy: [measured/theoretical concentrations] % (n ) 5). b Precision: % RSD (n )5). c Interday: All data from the 3 validation days are pooled (n )15).
used the microliter-pick up injection mode to automatically introduce IS to samples on-line. The process of the on-line addition of IS is described in detail in the Experimental Section and schemed in Figure 2. Briefly, the analyte and the IS are introduced on-line by the autosampler into the sample injection loop from the sample vial and a microreservoir containing the IS solution, respectively. As a result, the sample in the injection loop will be sandwiched between leading and tailing plugs of IS before being loaded onto the analytical column. On-Line versus Off-Line IS Methods. On-line SPE LC-MS/ MS methods with off-line and on-line introduction of IS were developed for model compounds (i.e., propranolol with ketoconazole as IS and diclofenac with ibuprofen as IS). Figure 3 shows representative chromatograms of propranolol/ketoconazole and diclofenac/ibuprofen samples in rat plasma with IS added on-line and off-line. The retention time and the peak widths of the analytes and IS peaks were identical for on-line and off-line introductions of the IS. Note that the sample volume injected onto the SPE cartridge with the off-line method is 10 µL, whereas with on-line IS introduction, 47.5 µL was loaded onto the SPE cartridge (10 µL of sample + 37.5 µL of IS). This needs to be taken into consideration when IS concentration is selected. To verify the reproducibility and repeatability of the amount/ volume of IS injected, the peak area of IS was monitored during 50 consecutive injections of plasma samples with IS added online and off-line (Figure 4). The variations (RSD) in IS peak area of samples spiked with IS off-line were 10.1% for ketoconazole and 2.1% for ibuprofen, respectively. However, when IS was introduced
Figure 4. IS signal intensity vs number of injections obtained in plasma samples for (A) ketaconazole (500 ng/mL) obtained in the positive ion mode and (B) ibuprofen (500 ng/mL) obtained in the negative ion mode.
on-line by the autosampler, variations in IS peak areas were 6.8% for ketoconazole and 3.1% for ibuprofen, respectively. Therefore, the on-line addition of IS using the autosampler provides reproducibility in the amount/volume of IS injected comparable to that obtained using off-line IS introduction (spiking samples manually using a pipet). Individual absolute recoveries of both propranolol, diclofenac, and their internal standards were found to be higher than 90% and were not significantly affected by the approach of IS introduction. Therefore, the extraction efficiency of the SPE cartridges Analytical Chemistry, Vol. 78, No. 4, February 15, 2006
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and the ionization suppression/enhancement were similar between the two methods despite the difference in the total volume loaded onto the SPE cartridge. The quality of quantification obtained using off-line and online IS introduction methods was studied using precision and accuracy as the validation criteria. Table 2 shows validation results obtained for propranolol and diclofenac. Analysis was performed directly from rat plasma using on-line SPE LC-MS/MS with IS added on-line and off-line. Off-line methods for both propranolol and diclofenac yielded accuracy in the range of 86-106% and precision ranging from 2 to 12%. On-line methods provided accuracy and precision in the range of 85-119% and 2-16%, respectively. Therefore, this new approach of adding IS on-line did not compromise the validation criteria of the developed methods by providing comparable accuracy and precision levels and can substitute the traditional approach of adding IS off-line. Furthermore, there were no practical difficulties encountered during sample analysis such as carryover or system contamination with the new technique. It is important to note that this new approach allows for the use of the autosampler as a measuring device to measure and introduce both sample (analyte) and IS. More than one internal standard can be potentially used if needed to monitor multiple analytes. With this approach, it is also possible to eliminate two off-line (manual) sample preparation steps, i.e., measuring fixed amount of samples and spiking with fixed amount of IS. Therefore, no off-line sample preparation or sample manipulation (e.g., aliquoting) prior to the on-line SPE LC-MS/MS analysis would be required whatsoever. Furthermore, this methodology allows for nonvolumetric sample (plasma) collection, and the entire process could potentially be fully automated. Due to low sample consumption (10 µL of plasma), and lack of any off-line sample manipulation, this approach can effectively be used for analysis of small-volume samples (e.g., mouse plasma) and analytes prone to nonspecific binding. It is important to note that other autosampler technical schemes and modes can be used to automatically introduce IS on-line. For example, analyte and IS can sequentially be aspirated
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by injector needle from two different vials (i.e., sample vial and IS vial). In that case, cross contamination between the two vials is a potential problem. Depending on the capabilities and the design of the autosampler, other approaches might be viable as well to combine and introduce sample and IS on-line. CONCLUSION A novel, simple, and elegant approach for on-line introduction of IS for quantitative analysis using LC-MS/MS has been developed. Methodology allowed to reliably introduce IS and demonstrated injection accuracy and precision comparable to those obtained using off-line IS introduction while maintaining chromatographic parameters. The assays based on this methodology yielded accuracy (85-119%), precision (2-16%), and analyte recovery comparable to those obtained using off-line IS introduction. Furthermore, on-line IS introduction allows for nonvolumetric sample (plasma) collection and direct analysis without the need of measuring and aliquoting a fixed sample volume prior to the on-line SPE LC-MS/MS analysis. Therefore, combination of the on-line addition of IS with on-line SPE sample preparation provides a powerful and fully automatic way for quantitative analyses of biologically active compounds in complex matrices. ACKNOWLEDGMENT This work was sponsored by 2003 and 2004 Pfizer students’ internship program. The authors thank Spark Holland, Inc. and in particular Rob Caspien, for providing the Symbiosis Pharma system for evaluation. Special thanks go to Steven Eendhuizen, Dirk Hiemstra, and Emile Koster of Spark Holland and John Lawrence of Pfizer for their technical assistance. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review October 9, 2005. Accepted December 14, 2005. AC051806Q
