Report
Sample Preparation for LC/MS/MS:
T
he issues and strategies that have historically been important in biological sample preparation and analysis remain fundamentally important to reliable LC/MS/MS. Some reports, however, have suggested that LC/MS/MS techniques do not require as much sample preparation or perhaps even none! Although the latter would be welcome to many analysts, life is usually not that simple. This Report reviews those issues that relate to the preparation of biological and environmental samples for LC/MS/MS. Currently, the most popular approaches to LC/MS/MS involve the atmospheric pressure ionization (API) techniques of electrospray and atmospheric pressure chemical ionization (APCI) (1-3). Although API mass spectrometers have greatly facilitated LC/MS applications to real-world problems (2), appropriate sample preparation combined with effective chromatography usually still the best analytical results (4)) Unfortunately combining an HPLC instrument with amass spectrometer could be considered an "unnatural marriage" As depicted by Arpino and Guiochon in 1979 (5) LC/MS combines an instrument that operates in the condensed phase with an
instrument that operates under vacuum The marriage of these two analytical techniques must take place while still allowing each techninue "to do its thing" Because of the two ery different regimes under which each instrument operates, care must 1,2
Jack Henion Edward Brewer 1 Geoffrey Rule 1
1
Advanced BioAnalytical Services and 2 Cornell University 650 A
Knowing the basicrequirementsand the big picture of an LC/MS system can ensure success in most instances. be taken in the way an integrated LC/MS system is used. Although several requirements must be met for each "partner" in the LC/MS marriage, perhaps only one major issue associated with each component of an LC/MS system is crucial. For the HPLC system, it is the mobile phase (and any modifiers) and flow rate that affect a chromatographic separation. For the API mass spectrometer, it is the mode of ionization and those factors that affect the production and transmission of gas-phase ions into the system. A successful LC/MS method will provide reproducible highly sensitive performance for hundreds of sample extracts on a daily basis A myriad of choices must be made however when considering the samplepreparation LC/MS This Report cannot address all the issues for successful biological and environmental sample preparation for LC/MS applications. We believe, however, that attention to certain important issues as well as a knowledge of the basic requirements and the big picture of an LC/MS system can ensure success in most instances. Several approaches to sample preparation that favor successful trace analysis will be covered with these principles in mind. The need for sample preparation prior to most biological and environmental analyses has long been realized. Liquid-liquid extrac-
Analytical Chemistry News & Features, October 1, 1998
tion has a long history and, although other techniques are available that have supplanted it in some cases, this technique still has value. More recently, liquid-solid extraction or, as it is more often called, solid-phase extraction (SPE), has grown in importance. Most current applications of sample preparation for LC/MS use some variant of these techniques (6-8). (A recent review covers many of the traditional extraction techniques [9].. We do noo intend to cover in netail all the issues that pertain to applying and optimizing these techniques. Rather we choose to discuss the relative merits of each as they apply to sample preparation for LC/MS analysis of biological and environmental samples Another important sample-preparation techniaue is supercritical fluid extraction (SFE) which has been in biolorical and in nnrtiriilar e n d r o n m e n
tal samnle nrenaration SFF is not covered in article in nart hecause of its limitations in handling a lartrp nirmber nf samples in a
fully automated fashion (10).
"Dilute and shoot" The ideal approach to sample preparation is to exclude the step altogether or "dilute and shoot". Some researchers advocate and have even demonstrated satisfactory LC/MS/MS analyses without any sample preparation. This approach is sometimes possible when sample levels of targeted
Analyzing Biological and Environmental Samples analytes. Alternatively, APCI has been shown to be less affected by this problem, so sometimes the dilute-and-shoot approach can be used if other important criteria are met (4,11,12). Liquid-liquid extraction
Liquid-liquid extraction usually involves mixing an aqueous sample solution with an equal volume of immiscible organic solvent for a period, allowing the two immiscible liquid phases to interact with the intent that the analyte(s) will be extracted from the aqueous layer into the organic layer. In some cases, as many as three immiscible layers may form because of the different densities of the solvents (13). Many factors affect the recovery and selectivity of the analytes from the aqueous solution, including analyte solubility and pKa, solution pH, and ionic strength. After separation of the immiscible liquids by centrifugation the organic layer containing the extracted analytes is removed concentrated to dryness and reconstituted in an appropriate solvent foreferably the HPLC mobile phase) for LC/MS analysis at least replicate extractions have been performed analytes are relatively high and the matrix components do not co-elute or otherwise interfere with ionization of the analytes. In a related strategy, proteins are sometimes precipitated by diluting plasma with excess acetonitrile, centrifuging the sample, and directly injecting an aliquot of the supernatent into the instrument. When an analysis is performed in this manner, using a very short HPLC column or no column at all, there are potential risks. Matrix-suppression of ionization can be particularly problematic under electro-
spray ionization conditions. It is generally known that this mode of ionization is sensitive to these effects (4). Therefore, practitioners should use postextraction spikedmatrix blanks and compare those results with analytical standards to demonstrate that such matrix effects are not interfering with the analysis. A matrix blank is a representative biological or environmental control sample that is known to be free of the targeted analytes. A spiked matrix blank is a control sample that has been fortified at defined relevant levels with die targeted
and the extracts combined More commonlytnHav a sincde e traction is carried t-i-
J
J
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• i
out to save time and material. One of the benefits of liquid-liquid extraction is that, with a judicious choice of solvent(s) and pH, very clean extracts can be obtained with good selectivity for the targeted analytes. The technique often uses large volumes of costly solvents, however, causing disposal problems. The procedure is also not amenable to automation because several disjointed steps are usually required. With today's need for parallel prepara-
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Figure 1 . Ion current profiles for 16-hydroxyestrone. Profiles were taken during (left to right) the 1st (well #17), 11th (well #174), and 23rd (well #366) hour of continuous LC/MS/MS analysis. Each trace shown was from the low QC (8 ng/mL) sample and was acquired using positive ion APCI LC/MS/MS conditions to monitor the transition mlz 287.2 to 198.9.
tion of hundreds of samples per day, this technique becomes a significant bottleneck. Because the extracts are relatively clean and the selectivity is relatively good, however, hundreds of liquid-extracted samples may be run on an LC/MS system with good sensitivity and without degradation of system performance. It is for these reasons that analytical chemists need to develop robotic, parallel, liquid-liquid extraction devices for large numbers of samples. One problem in liquid-liquid extraction is minimizing exposure of the analytes to potentially adsorptive surfaces. Exposing analytes to silanol groups on silica surfaces found in solid-phase extraction techniques can reduce recovery. This result is less likely during liquid-liquid extraction, but adsorptive losses may occur during the solvent removal or "blowdown" step following extraction, when the analyte is allowed to form a dry residue on the inside surface of the container. In addition aerosol ation and/or evaporative losses during the blowdown step if the analyte forms particulates or is relatively volatile Although impractical not concentrating the extraction solvent layer to dryness can 652 A
obviate these potential problems. The final reconstitution solvent should dissolve the extract completely and be compatible with the initial HPLC mobile phase. When all of these conditions are met, liquid-liquid extraction provides excellent results.
cinnamine, was used as the internal standard. The reconstituted extracts were analyzed by ion spray LC/MS/MS techniques in the positive ion-selected reaction monitoring (SRM) mode. The calibration curve for reserpine extracted from equine plasma using liquid-liquid extraction was linear from 10-5000 pg/mL and, using SPE, ,i was linear from 100-5000 pg/mL The limit of quantitation using liquid-liquid extraction and SPE was 50 pg/mL and 200 pg/mL, respectively. The limit of detection for reserpine by LC/ MS/MS was 10 pg/mL Recent adaptations of SPE have appeared that are well-suited for LC/MS applications, including using SPE sample preparation in the 96-well format (14). Several related formats have recently appeared using miniaturized packed-bed formats as well as an adaptation of disk technology for the 96-well format. Our goal for the 96-well approach is to add a higher level of parallel sample preparation using essentially a further miniaturized SPE format. By extracting multiple 96-well format SPE plates up to four 96-well plates or 384 samples may be prepared and then analyzed within 24 hours by and LC/MS
tern which represents a significant improvement in sample throughput commercially available equipment and supplies Future developments should further increase throiighput significantlv Figure 1 shows representative LC/ MS/MS results using 96-well SPE for the Solid-phase extraction SPE has its commercial roots in the late 70ss preparation and analysis of 16-hydroxyestrone in urine. The figure shows three Since then, it has become a common and SRM ion current profiles for samples run effective technique for extracting analytes from complex samples. SPE prepares multi- during the 1st, 11th, and 23rd hours. The four 96-well sample blocks, which contained ple samples in parallel (typically 12-24) and uses relatively low quantities of solvents, and the final eluate from the SPE extraction procedure, were placed in an autosampler. The the procedures can be readily automated. isocratic HPLC run time for each sample Although the overall benefits of SPE for was 3.6 min, allowing more than 384 4amLC/MS applications are positive, extracts are often rather dirty and may contain fines ples to be analyzed within a 23-h period. or small particulates that can cause pressure There are some important issues rebuildup in HPLC columns. Our experience garding development and validation of also suggests that using SPE may result in methods using this procedure, including reduced trace-level recoveries for the stability of the analytes as they sit in pounds that are particularly adsorptive to the autosampler, maintaining LC/MS senactive surfaces such as basic drugs sitivity and ruggedness over extended periods and processing and handling large volAn example of a comparison between umes of data. In spite of these issues, this liquid-liquid extraction and SPE is the approach offers a significant improvement quantitative determination of reserpine in equine plasma {&). A structural analog, res- in high sample-throughput analysis. Note
Analytical Chemistry News & Features, October 1, 1998
in Figure 1 that the retention times and ion current abundances noted as "cps" at the top of each chromatogram remained constant through this 23-hour period. Affinity techniques coupled w i t h LC/MS
A high degree of molecular selectivity can be achieved with affinity chromatography and affinity extractions (15). These techniques are based upon molecular recognition and can isolate and concentrate specific analytes, or classes of analytes, from complex sample matrices. With this approach, a high degree of purification of a targeted analyte is possible in a single step from large volumes of sample. A practical example of this approach is the use of affinity chromatography to selectively capture pesticides or herbicides from
environmental samples for quantitation at the part-per-trillion level (16-18). Sensitive assays for these compounds have been developed, which rely on the ability of an immobilized antibody column to concentrate analytes from dilute samples (19-22). One limitation of affinity chromatography, however, is the restricted capacity and the labile nature of covalently bound protein receptors, which can cause problems for reliable quantitative analysis. Combining immunoaffinity extraction (IAE) (19,23) and ultrafiltration (24,25) with MS could provide an effective approach to analyzing complex samples by exploiting the advantages offered by each technique. A practical strategy is to couple IAE on-llne with LC/MS (22,23) or to prepare samples off-line using ultrafiltration combined with column-switching techniques (21). IAE mini-
mizes sample preparation procedures and selectively concentrates analytes, and LC/MS provides a rapid and very selecttve detection method for captured analytes when eluted from the affinity medium. When IAE techniques are used, analytes from a sample matrix may be detected by LC/MS/MS monitoring of selected precursor and product ion pairs to provide sensitivity sufficient for ultratrace quantitation. For example, the illegal drug LSD and iis metab- • olites were enriched from urine sample volumes diluted with phosphate-buffered saline (19). IAE has an advantage over other rorms of extraction when the sample matrix contains an excess of interfering components. In this example diluted human urine samples were passed through an immunoaffinirv HPLC column Affer directing the unretaitipH pflmponPTits to waste the "recnc-
Figure 2. Immunoaffinity extraction followed by column switching (LC/LC) and SUM LC/MS of (a) control blank urine and (b) an LSD-positive urine specimen. Analytical Chemistry News & Features, October 1, 1998 653 A
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nized" LSD and iis metabolites were released by denaturation of the antibody, whereupon the analytes passed directly to a chromatographic system coupled to a triple quadrupole mass spectrometer. It is important to note that when a single mass analyzer benchtop LC/MS system is operated in the selected ion-monitoring mode, chemical background noise at the selected m/z may cause some interference. The background can consist not only of residual chemical interference from the extracted sample but also interfering ion clusters from HPLC buffer components. SRM LC/MS experiments, however, provide an extra level of "filtering", which eliminates these sources of interference and produces significant gains in S/N and improved lyte specificitv In addition the polyclonal antibody immunoaffinity media captured LSD and its metabolites in the above studies IAE provided preconcentration but unwanted compounds were also captured via nonspecific binding. SRM I £ / M S detection provided additional separation hv mass selective detection of the Ivt
The final effluent is then directly coupled to an LC/MS system (26). The second approach uses robotic equipment to perform on-line SPE of crude samples, followed by LC/MS analysis. Column-switching
Column-switching, techniques afford an interesting and creative alternative for sample preparation. This approach depends on the selectivity of appropriately chosen HPLC stationary phases to retain and separate the analyte(s) of interest while allowing unretained components to be eliminated from the column. An additional HPLC column with differing selectivity may be placed in series to provide enhanced selectivity and separation of targeted analytes from matrix ccmponents. The preferred approach in our laboratory (20) involves a backwash of fhe second column after the targeted compound (s) have been trapped at the inlet of the second column. This second HPLC column acts as the "injector" of the trapped components onto a third column, which affords the final analyti-
cal HPLC separation and elution to the LC/MS system. The benefits afforded by this approach include total automation and quantitative transfer of targeted components within the column-switching system. In the "heartcut" mode (27), a narrow retention-time region containing a desired component(s) is "cut" from the chromatogram and transferred onto another HPLC column for further separation. In this instance, quantitative transfer of the components without adsorptive or degradative losses can be assumed. The other advantage of this approach includes the increased selectivity afforded by the judicious choice of two or HPLC stationary phases The limitations of this approach include restricted sample enrichment because of the limited amount of original, untreated, crude sample that may be loaded onto the first column of the HPLC separation. As a result, a column-switching method may not reach the lower limit of detection required for the problem at hand. Another limitation
If these concepts are applied to real samples, useful results may be obtained. Several LSD-positive human urine specimens were analyzed with IAE techniques, followed by column-switching (LC/LC) and SRM LC/MS techniques. A 100-mL sample consisting of 10% urine and 90% phosphatebuffered saline was pumped through a protein G column loaded with anti-LSD antibody at 4 mL/min. Figure 2(a) shows chromatograms obtained with a control blank urine, and Figure 2(b) shows an LSD-positive urine specimen. The original measured concentration of LSD in the specimen determined by GC/MS was 0 9 ng/mL The labeled peaks in Figure 2(b) indicate the presence of LSD and its metabolites These results demonstrate the practicality of online affinity techniques for sample cleanup
and trace enrichment of targeted comp n n n r l s in r n m p l p v
samples An alternative approach is robotic sample preparation on-line with real-time LC/MS analysis. At least two approaches deserve consideration. One approach uses direct injection of untreated sample onto a chromatographic system, followed by column-switching (LC/LC) sequences that selectively retain the analytes of interest. 654 A
Figure 3. On-line sample pretreatment prior to quantitative SRM LC/MS analysis of (a) proprietary drug and (b) its D5 internal standard.
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is that sample throughput using this approach will likely not be as high as for other methods. Instead of 2-5-min run times, a column-switching method may require 6-12 min or more to affect an individual sample cleanup and LC/MS analysis. Finally, some analysts believe that setup and use of a column-switching method is too complicated to be practical. We think that this is an unfortunate misunderstanding because modern instruments make these procedures relatively straightforward. One example of a coupled-column LC/ MS/MS analysis of an environmental sample that also includes an IAE component is the isolation of carbendazim from soil extracts (21). The data were obtained by a method that uses a high-performance protein G immunoaffinity column coupled to a reversed-phase analytical column by a trapping column. The specificity of IAE makes it possible to detect low levels of carbendazim in soil samples without interference from matrix components. The column-switching components of this system allow "decoupling" of the high ionic strength buffers needed for IAE from the electrospray LC/MS system. The high salt content, which may co-elute with sample components of interest, can be very detrimental to electrospray detection of targeted organic compounds (4). In addition, column-switching allows some additional selectivity for isolating the targeted analyte from the analyte and antibody released from the IAE process SR1VI vides good sensitivity with a minimum of chemical interference Carbendazim was enriched from soil extracts in this instance by IAE at the 100 ppblevel. It is also possible to detect trace levels (25 pptr) of carbendazim in lake water using this technique (21). This improved limit of detection over the soil analysis is probably caused by fewer matrix interferences in water than in soil. Very little sample preparation is required for environmental water samples, whereas a liquid-solid extraction of the soil is required prior to IAE of soil samples. Thii method allows for the analysis of multiple samples in a relatively short period with minimum sample preparation With the described protocol three soil or water samples can be analyzed per hour without operator intervention
On-line SPE Another approach is on-line SPE for the automated preparation of samples prior to LC/MS analysis (28,29). This approach uses a commercial device that combines an autosampler and a solvent delivery unit to aliquot multiple liquid samples into a flowing stream of solvent. The solvent has preconditioned an in-line SPE cartridge. After conditioning, the SPE cartridge retains the targeted analytes while the relatively weak solvent elutes unretained salts and polar matrix components to waste. An empirically optimized sequence of increasingly stronger solvents is then used to further elute weaklv retained unwanted sample components A final elution with HPLC mobile phase elutes the targeted analytes
LC/MS concepts will be pivotal, and these systems will likely be more common than GC/MS. off the SPE cartridge and onto an analytical HPLC column for LC/MS analysis. Because this particular autosampler may be loaded with up to 160 samples, an entire tray of samples may be automatically prepared and analyzed by LC/MS in an unattended sequential fashion. A sample is always being analyzed by LC/MS while the next sample is being prepped. The advantages of this sequential automated system include analyte trace enrichment, unattended on-line sample preparation and analysis, and minimized adsorptive losses that often occur with off-line sample transfers and sample-handling procedures. Limitations include those aspects typical of any serial analysis, such as reduced sample throughput and sample stability problems
caused by extended storage times in the autosampler. It should be noted that in one report (28), improved MS/MS sample throughput was demonstrated by excluding the analytical HPLC column, which effectively provided automated on-line flowinjection analysis of the SPE extracts. The total automation features of this approach and impressive detection limits that may be achieved make this sample-preparation strategy desirable in certain instances. Figure 3 shows SRM LC/MS ion currrnt profiles obtained from the on-line SPE isolation of a proprietary drug and its D5 internal standard isolated from 250 uL of plasma using the system described above. Figure 3(a) shows the ion current profile for the parent drug at 50 pg/mL in plasma. Figure 3 (b) shows the corresponding ion-current profile for the D5 internal standard at 10 ng/mL. .n this experiment, 0.2 mL of buffer-diluted plasma (1:1) was loaded onto the SPE cartridge and extracted robotically on-line with concomitant LC/MS/MS analysis of each sequential sample The LC/MS experiment carried out using a 2 mm i d x x5 mm Zorbax SBCN column maintained at a flow of 0 2 mL/min with 80/20 acetonitrilee10 mM ammonium arptatp r»H 4fimnhilp nha«p This approach provided sample throughout of 12 samoles/h This on-line SPF annroarh often provides improved detection limits over related offline pro d th t sample transfers and sample concentration procedures. Down the road The need for some degree of sample preparation prior to LC/MS analysis will llkely continue. Current trends appear to focus on minor improvements to existing technologies. The manner in which we approach this topic in the future, however, should continually be evaluated. Allhough there is a move toward parallel sample processing and miniaturization, more dramatic changes will likely be required for the future. We believe the sample preparation and analysis scheme of the future will include microdevices designed for nanoliter quantities of sample in chip-based reservoirs connected via nanofabricated closed channels. These channels will be integrated with miniaturized formats that will include HPLC and electrophoretic separations. We
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Report believe the effluent from these separations will be directly connected to a preferred detector, probably a mass spectrometer. This miniaturized sample preparation, separation, and analysis system will be an integrated unit, making our current concepts appear very old-fashioned. Miniaturization affords challenges and benefits. The application of nanoliter quantities of samples and materials to chip-based analysis systems will require nanopipetters capable of dispensing large numbers of samples in parallel. The nanofabricated devices will be very inexpensive and disposable and will preclude current problems associated with contamination or carryover between samples because each sample track will be used only once. This strategy will also demand continued improvements in LC/MS sensitivity because very small quantities of sample will be used Although this may seem a challenge modern techniques often use only 50 uL of plasma for example whereas 10 years ago 5-10 mL of biological samrile were IIQPH
A final point to consider is the increased demand for much higher sample throughput than is provided today. Typically, an LC/MS experiment involves serial analysis of an autosampler tray with sample throughput of up to 384 samples per 24-h period. The analytical demands of modern drug discovery practices, such as drug mixture dosing to single animals and analysis of samples from high-throughput screening of combinatorial chemical syntheses that generate very large numbers of samples will demand much improved sample preparation and analysis capabilities We believe one key to improving sample throughput is to prepare and analyze samples in parallel. The so-called "massively parallel" concept imposes some interesting challenges to the way things are currently done. A massively parallel system has two orthogonal parallel axes in simultaneous operation (30). We envision technologies that will allow simultaneous, parallel sample preparation and chromatographic separation and detection of for example 96 samples. Thus, multiples of the current 96-well plates may be miniaturized to integrated analysis formats Our vision would allow the simultaneous parallel preparation of 96 biological samples which could be transferred directly 656 A
and simultaneously to 96 HPLC or electrophoretic separation channels. After simultaneous chromatographic separation of the 96 sample extracts, the effluent from these separations would be transferred directly to a mass spectrometer, which could monitor the effluent from all 96 columns simultaneously. Thus, such an instrument, which does not currently exist commercially, would have 96 parallel ion paths or mass analyzers in one vacuum system (30). Detection of parallel ion beams and data handling of 96 separate signals would impose new dcrnsnds on the ixiciss SDectrometer data system but these obstacles must be overOf course the high volume of data generated by this anDroach will also challenge information technolocrv and qualityassuranrp programs The goal will be to develop intetrrated miniaturized chip-based devirps that form ni rni i tine* T f~* /1V/TQ
we come In pvnprt from mnHprn instrumentation. Automation and integration of everything from sample preparation to detection and data analysis will be important components of future developments in miniaturized LC/MS systems. Parallel concepts should be used whenever possible because so much more may be accomplished in this mode. The future is bright for technique and instrumentation developments that can revolutionize the way we currently approach our analytical problems. LC/MS concepts will be pivotal to future developments and these systems will likely be even more common than GC/MS instruments are today References (1) Bruins, A. P. Trends Anal. Chem. 1994, 13,37-43. (2) Bruins, A. P. Trends Anal. Chem. 1994, 13, 81-90. (3) Brewer, E. A; Henion, J. D. /. Pharm. Sci. 1998,87,395-402. (4) Matuszewski, B. K.; Constanzer, M. L.; Chavez-Eng, C. U.Anal. Chem. 1998, 70, 882-89. (5) Arpino, P. J.; Guiochon, G.J. .hromatogr. 1979,185, 529-47. (6) Anderson, M. A; Wachs, T.. Henion, ,J D. /. Mass Spectrom. 1997,32,152-58. (7) Wu, Y.; Lii L Y-T.; Henion, J. D.J. Mass Spectrom. .196,31,987-93. (8) Wu, Y; Zhao, J.; Henion, J.; Korfmacher, W. A; Lapiquera, M. P.; Lin, C-C.J. Mass Spectrom. .197,32,379-87. (9) Majors, R. E. LC-GC Supplement 1998, May, S8-S15.
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(10) Taylor, L. Supercritical Fluid Extraction; John Wiley & Sons: New York, ,196. (11) Covey, T. R.; Lee, E. D.. Bruins, A P.; Henion, J. D.Anal. Chem. .186,58, 1451A-61 A. (12) Schaefer, W. H.; Dixon, Jr.. F.J. Am. .oc. Mass Spectrom. 1996, 6,1059-69(13) Covey, T. R.; Silvestre, D.; Hoffman, M. K.; Henion, J. D. Biomed. and Environ. Mass Spectrom. .988,15,45-56. (14) Allanson, J. P.; Biddlecomebe, R. A; Jones, A E.; Pleasance, S. Rapid Coom. Mass Spectrom. .996,10,811-16. (15) Larsen, B. S.; McEwen, C. N. Mass Spectrometry of Biological Materials, 2nd. ed.; Marcel Dekker: New York, 1998. 81-98. (16) Slobodnik, J. H. et al./ Chromatogr. A. 1996, 741,59-74. (17) Slobodnik, J.; van Baar, B.L.M.; Brinkmaa, VAT.J. Chromatogr. A 1995, 703,81-121. (18) Muelenberg, E. P.; Mulder, W. H.; Stoks, P. G. Environ. .ci. Tech. 1994,29,533-61. (19) Cai,J.;Henion,J. D.Anal. Chem. .996, 68, 72-78. (20) Cai, J.; Henion, J. D.J. Chromatogr. B. 1997, 691,357-70. (21) Bean, K. A; Henion, J. D.J. Chromatogr. A. 1997, 791,119-26. (22) Rule, G. S.; Mordehai, A V.; Henion, ,J Anal. Chem. 1194, 66,230-35. (23) Nedved, M. L; Habbi-Goudarzi, S.; Ganem, B.; Henion, J. D. Anal. Chem. .196, 68, 4228-36. (24) Wieboldt, R; Zweigenbaum, J.; Henion, J. Anal. Chem. 1197, 69,1683-91. (25) van Breemen, R. B.; Huang, C-R; Nikolic, D.; Woodbury, C. P.; Zhao, Y-Z.; Venton, D. L Anal. Chem. 1997, 69,2159-64. (26) van der Hoeven, RAM. et al./ Chromatogr. .997, ,62,193-200. (27) Edlund, P. O.; Bowers, L; Henion, J. D. / Chromatogr. 1989,487,341-56. (28) Bowers, G. D.; Clegg, C. P.; Hughes, S. C; Harker, A J.; Lambert, S. LC-GC 1997, 15,48-53. (29) McLoughlin, D. A; Olah, T. V.; Gilbert, J. D.J. Pharm. and Biomed. Anal. 1997, 15,1803-1901. (30) Kirchner, N. J. US Patent No. 5,206,506 1993. Jack Henion is president and CEO oo Advanced BioAnalytical Services (ABS) )nd professor of toxicology at Cornell University. His research interests include advancing technology for efficient automated sample preparation with coupled separation sciences and MS. Edward Brewer is sectton head for rrug giscovery at ABS. His research interests include integrating automated sample preparation procedures wiih HPLC/MS/MS techniquess Geoffrey S. Rule is a senior research scientist at ABS. His research interests include dedelopment of novel strategies for rapid determination ofdrugs toxins and pathogenic organisms in biological matrices using LC/MS/MS techniques Address correspondence to Henion at ABS 15 Catherwood Road,thaca NY 14850 (jhenion@abs-lcms com)
