Anal. Chem. 1999, 71, 4107-4110
Postcolumn Introduction of an Internal Standard for Quantitative LC-MS Analysis Bernard K. Choi,† Arkady I. Gusev,‡ and David M. Hercules*,†
Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37235, and Rohm and Haas, Philadelphia, Pennsylvania 19106
A method for introducing internal standards by direct infusion into the LC effluent for the quantitative LC-MS analysis of environmental samples is described. This postcolumn introduction method was found to be effective in correcting quantitative errors associated with matrix signal suppression. However, unlike surrogate or volumetric internal standards, the performance of the postcolumn method does not depend on the selection of an internal standard that shares identical elution time with the target analyte. As a result, either structural analogues (target analyte derivatives) or isotopically labeled compounds may be applied as effective internal standards. Furthermore, the postcolumn introduction method allows the application of one internal standard to address signal suppression effects for several analytes in a single LCMS run. In contrast, volumetric and surrogate introduction methods require an isotopically labeled internal standard for each analyte to be quantified. Electrospray mass spectrometry (ESI-MS) is an established instrumental technique for the analysis of fragile nonvolatile organic compounds.1,2 This technique is widely used as an online detector for liquid chromatography (LC). The separating power of LC combined with the sensitivity and selectivity of ESIMS has allowed liquid chromatography-mass spectrometry (LCMS) to develop into a standard analytical technique in the areas of bioorganic and environmental chemistry.3,4 Due to the stable nature of the ion source, ESI-MS is one of the few mass spectrometry techniques capable of performing quantitative analysis using external standards (i.e., calibration using absolute signal response). However, ESI-MS is prone to signal suppression or enhancement effects when samples extracted from physiological or environmental matrixes are analyzed.5-9 One technique that can be used to compensate for these matrix signal effects is the application of internal standards.10 There are generally two methods by which the internal standard is introduced into the sample. The surrogate introduction method involves addition of the internal standard prior to any †
Vanderbilt University. ‡ Rohm and Haas. (1) Balogh, M. P. LC GC-Mag. Sep. Sci. 1998, 16, 135-140. (2) Niessen, W. M. A.; Tinke, A. P. J. Chromatogr., A 1995, 703, 37-57. (3) Ferrer, I.; Barcelo, D. Analusis 1998, 26, M118-M112. (4) Slobodnik, J.; Van Barr, B. L. M.; Brinkman, U. A. Th. J. Chromatogr., A 1995, 703, 81-121. 10.1021/ac990312o CCC: $18.00 Published on Web 08/07/1999
© 1999 American Chemical Society
Figure 1. Structures of compounds used in this study.
extraction procedure.10 This internal standard compensates for signal loss attributed to sample preparation (e.g., extraction, purification). In contrast, the volumetric introduction method involves the addition of internal standards immediately prior to analysis10 to address instrumental errors (e.g., injection volumes, fluctuations in the ionization process). Both surrogate and volumetric introduction methods can be used to compensate for quantitative errors attributed to signal suppression in LC-MS. However, there are limitations associated with these methods. For either method to be effective, the analyte and the internal standard must elute from the LC column simultaneously. Simultaneous elution ensures that ionization of both compounds occurs under identical conditions. For this reason, isotopically labeled compounds are generally required for use as volumetric and surrogate internal standards. As a consequence, analysis of multicomponent mixtures generally requires an isotopically labeled internal standard for each analyte to be quantified. Due to difficulties associated with synthesis and/or cost, isotopically labeled internal standards may not be readily available for some analytes. However, another internal standard introduction method may be used which would ensure the simultaneous (5) Foltz, R. L.; Edom, R. W. J. Mass Spectrom. Soc. Jpn. 1998, 46, 235-239. (6) Buhrman, D. L.; Price, P. I.; Rudewicz, P. J. J. Am. Soc. Mass Spectrom. 1996, 7, 1099-1105. (7) Kebarle, P.; Tang, L. Anal. Chem. 1993, 65, 972A-986A. (8) Mirza, U. A.; Chait, B. T. Anal. Chem. 1994, 66, 2898-2904. (9) Yen, T. Y.; Chalres, M. J.; Voyksner, R. D. J. Am. Soc. Mass Spectrom. 1996, 7, 1106-1108. (10) Boyd, R. K. Rapid Commun. Mass Spectrom. 1993, 7, 257-271.
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Figure 2. Schematic of postcolumn introduction method for internal standards.
Figure 3. (A) Mass spectra of tebufenozide (1.2 ppm) with [13C]tebufenozide introduced by postcolumn infusion. Single-ion chromatographs of (B) tebufenozide and (C) [13C]tebufenozide internal standard.
ionization of the target analyte and the internal standard, regardless of analyte elution time. This alternative method involves infusion of the internal standard into the LC effluent. In this note, the postcolumn method for introducing an internal standard to address LC-MS signal suppression is described. [13C]tebufenozide was introduced as an internal standard by direct infusion into the LC effluent during LC-MS analysis of tebufenozide and hydroxytebufenozide in a wheat hay matrix (Figure 1). Application of this introduction method for the analysis of singlecomponent and multicomponent mixtures will be discussed. EXPERIMENTAL SECTION Materials. Tebufenozide (active ingredient in Mimic, and Confirm insecticides), hydroxytebufenozide, and [13C]tebufenozide 4108 Analytical Chemistry, Vol. 71, No. 18, September 15, 1999
(Figure 1) were obtained from the Rohm and Haas Co. (Philadelphia, PA). Solvents used in the extraction and cleanup procedure were of HPLC grade and obtained from Fisher Scientific (Pittsburgh, PA). Sample Preparation. Preparation of the wheat hay extract involved the following. Two grams of wheat hay was mixed with 150 mL of an extraction solvent (90% methanol/10% 0.10 N HCl) and shaken for ∼30 min. The extract was transferred to a 500mL separatory funnel for liquid-liquid extraction with hexane and dichloromethane. Hexane partition involved the addition of 20 mL of 20% NaCl aqueous solution followed by 50 mL of 100% n-hexane. The aqueous phase was saved for dichloromethane partition. A 100-mL aliquot of 20% aqueous NaCl was added followed by 100 mL of dichloromethane. The organic phase was collected, followed by a second 100-mL dichloromethane partition. Both fractions of the dichloromethane organic phase were combined and evaporated using a rotary evaporator. The sample was redissolved in 4 mL of 1:1 acetonitrile/water and spiked with a known amount of tebufenozide and hydroxybufenozide (matrix sample set). A standard solution set was prepared by spiking known amounts of the analytes used in 1:1 acetonitrile/water. LC-MS Conditions. Liquid chromatography was carried out using a HPLC-1100 (Hewlett-Packard, Wilmington, DE); the injection volume was 100 µL. Analytes were chromatographed with a Supelco (Supelco, Bellefonte, PA) Supelcosil LC-18-DB (15 cm × 3 mm, 5 µm) column. The mobile phase was a gradient mixture of 0.1% formic acid in water (phase A) and 0.1% formic acid in acetonitrile (phase B). The flow rate was 0.8 mL/min. The initial gradient was 60% phase A and was decreased to 40% at time 1 min. Phase A was decreased to 10% at time 6 min. Phase A was returned back to 60% at time 8 min. LC-MS analysis was performed on a Mariner electrospray time-of-flight mass spectrometer (Perseptive Biosystems, Framingham, MA). Effluent from the LC was split to allow a flow rate of ∼10 µL/min to be infused into the ion source. A syringe pump (Cole-Parmer Instrument Co., Vernon Hills, IL) was used to mix 1.0 ppm [13C]tebufenozide into the LC/effluent, immediately prior to the ion source, at a flow rate of ∼1 µL/min. A diagram of the
Figure 4. Calibration curves for tebufenozide and hydroxytebufenozide using external standard signal calibration (A, B), volumetric internal standards (C, D), and [13C]tebufenozide postcolumn internal standard (E, F).
experimental arrangement is shown in Figure 2. The splitting and mixing were accomplished by Tee connection fittings. Mass spectra were acquired for 2 s for the mass range 340-380 m/z. Three sets of runs were performed in sequence by LC: a standard solution set, a matrix sample set, and a repeat of the standard solution set. RESULTS AND DISCUSSION The postcolumn introduction method involves the direct infusion of an internal standard into the LC effluent, as shown in Figure 2. Continuous infusion at a fixed flow rate allows the internal standard to volumetrically mix with all components eluting from the LC column. This ensures that the target analyte and the selected internal standard will ionize under identical conditions, regardless of the analyte’s elution time. Figure 3 shows the mass spectra acquired during the LCelution of tebufenozide while [13C]tebufenozide is introduced by postcolumn infusion. The signal characteristic of tebufenozide is the 12C peak. Although containing a contribution in part from the naturally occurring 13C isotopic species of tebufenozide, the 13C peak is primarily characteristic of 13C tebufenozide. Panels B and C of Figure 3 show the single-ion chromatographs of tebufenozide and [13C]tebufenozide, respectively. By taking the ratio of signal heights from the target analyte and internal standard, a relative analyte response, compensated for matrix signal suppression, can be obtained. Standard solutions and matrix sample sets of tebufenozide and hydroxytebufenozide were analyzed by LC-MS using [13C]tebufenozide as an internal standard. Three sets of LC-MS analysis runs were performed in sequence: a standard solution set, a matrix sample set, and a repeat of the standard solution set. Panels A and B of Figure 4 show the calibration curves for tebufenozide and hydroxytebufenozide, respectively using external standards (generating a calibration curve using absolute signal response). Consistently lower signal response was obtained from
the matrix sample set compared to that from the corresponding standard solution samples. A matrix memory effect (matrix signal effects carrying over to subsequent LC-MS sample runs) was also evident by a lower signal response obtained during the repeat of the standard solution set. The overall signal suppression effect ranged from 10 to 30%. Due to the extent of signal suppression, the application of an external standard for calibration is not possible for quantification of this system. Figure 4C,D shows calibration curves obtained using [13C]tebufenozide as a volumetric internal standard (internal standard is introduced prior to analysis). The use of [13C]tebufenozide as an internal standard provided signal compensation for tebufenozide, but not for hydroxytebufenozide. The [13C]tebufenozide internal standard elutes ∼3 min after the hydroxytebufenozide analyte. It is likely that the internal standard and target analyte are eluting with different matrix components and thereby experiencing different levels of signal suppression. This would account for the poor signal compensation of hydroxytebufenozide. Panels E and F of Figure 4 show calibration curves obtained for tebufenozide and hydroxytebufenozide, respectively, using postcolumn infused [13C]tebufenozide as an internal standard. The postcolumn internal standard method provided effective signal compensation for both tebufenozide and hydroxytebufenzoide. This shows that this method of introducing an internal standard does not necessarily require isotopically labeled internal standards to compensate for signal suppression. Particularly, this demonstrates that the postcolumn introduction method allows the application of a single internal standard to compensate signal suppression for multiple analytes. The postcolumn introduction method offers a number of advantages over conventional volumetric or surrogate introduction methods. Because this method allows structural analogues to be used as viable internal standards for LC-MS analysis, the postcolumn introduction method may find use in applications where isotopically labeled internal standards are not readily Analytical Chemistry, Vol. 71, No. 18, September 15, 1999
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available. LC-MS applications for the quantification of multicomponent sample mixtures can also be performed more efficiently by calibrating several analytes with a single internal standard. In addition, the quantity of the internal standard introduced may be readily controlled by adjustment of the infusion flow rate and/or solution concentration. Although the postcolumn introduction technique cannot account for signal loss attributed to sample preparation (e.g., extraction, cleanup), this method can be applied to a number of
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applications where the sample loss during the preparation procedure is minimal. This internal standard introduction method may also be useful in compensating for matrix signal effects in method development applications, where sample loss associated with the preparation procedures is evaluated. Received for review March 23, 1999. Accepted June 25, 1999. AC990312O
