A REVIEW OF CLINICAL DIAGNOSTIC APPLICATIONS OF LIQUID CHROMATOGRAPHYTANDEM MASS SPECTROMETRY

Bori Shushan* Clinical Mass Spec Consultants, Toronto, ON, Canada, M4W 2W6
Received 18 June 2009; accepted 8 September 2009
Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/mas.20295

Liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) technology is emerging as a complementary method to traditional methodology used for clinical applications. Enhanced specicity and high-throughput capabilities are providing signicant benets to clinical diagnostic laboratories conducting routine analyses. This technology is expected to expand rapidly as scientists focus on more complicated challenges that can be solved efciently by adding LC/MS/MS to their arsenal of techniques. # 2010 Wiley Periodicals, Inc., Mass Spec Rev 29:930944, 2010 Keywords: tandem mass spectrometry; clinical diagnosis; multidimensional chromatography; atmospheric pressure ionization; multiplexed multiparametric assays

I. INTRODUCTION
Despite some very early research on the use of mass spectrometry (MS) for respiratory gas analysis in the 1950s (Fowler & HughJones, 1957), the majority of early applications of MS in clinical diagnosis go back to the early 1970s with the application of GC/ MS to the determination of a variety of biologically signicant molecules. Because GC requires a certain level of analyte volatility and, since most biologically active molecules are polar, thermolabile, and involatile, elaborate extraction and derivatization protocols had to be devised to make GC/MS useful for the analysis of clinically relevant analytes and samples. To make sample analysis less difcult by MS there had been a signicant amount of R&D invested over several decades aimed at coupling HPLC with MS, since HPLC is a much better separation technology than GC for polar thermolabile biologically relevant molecules. This coupling was not without signicant challenges. Most of the LC/MS coupling techniques, which evolved over the 1970s and 1980s, were not very successful and many of those that enjoyed some widespread popularity such as Thermospray, Flow-FAB, and Particle Beam are now virtually extinct. All these techniques were quickly displaced by interfaces involving ionization techniques that separated the ionization process from the high vacuum analyzer portion of the instruments, namely, atmospheric pressure ionization (API) including electrospray (ESI), ion-spray (nebulizer-assisted electrospray, ISP), atmospheric pressure chemical ionization (APCI) using a heated nebulizer and atmospheric pressure photoionization (APPI) also using a heated nebulizer. With the development of API there was

This article is dedicated to the memory of Dr. William R. (Bill) Davidson, one of Canadas scientic leaders and an inspiration to many. *Correspondence to: Bori Shushan, Clinical Mass Spec Consultants, Toronto, ON, Canada, M4W 2W6. E-mail: bori@rogers.com

a much more rapid adoption of LC/MS for the analysis of clinically important samples. An exceptionally good review of the development of API techniques was written by Thomson (1998). A very recent account of the development of pneumatically assisted electrospray (a.k.a. IonSpray) and related techniques and their relevance to clinical diagnostics was just published by Henion (2009). As the name implies API creates ions at atmospheric pressure prior to and outside the pristine analyzer, thereby making LC/MS very robust and relatively free of analyzer contamination. Coupling of HPLC to API-MS is trivial since the MS vacuum system is not directly involved. This relative ease-ofuse permitted those researchers more familiar with biochemistry than with analytical technologies to actually use LC/MS more routinely. This in turn led to an enormous increase in applications of LC/MS across a wide range of biologically signicant molecules, including biopolymers like polysaccharides, DNA, and proteins as well as small molecule analytes such as nucleotides, amino acids, acylcarnitines, sphingolipids, phospholipids, biogenic amines, etc. The vast majority of LC/MS applications for clinical diagnosis today are being run using API. One of the important features of API techniques is that they are very soft, creating primarily intact ionized molecules (e.g., protonated, de-protonated, ammoniated, etc.) which, unless very high resolution and mass accuracies are employed, are not very analytically specic. This has necessitated the use of tandem mass spectrometry (MS/MS) to enable the analysis of trace analytes from complex biological matrices typically encountered in clinical diagnostic samples. There have been some excellent reviews on this subject to which the reader is referred (Dooley, 2003; Vogeser & Seger, 2008). Examples of the most prevalent uses of MS/MS in clinical diagnosis are: the screening of newborns for congenital metabolic diseases such as aminoacidopathies, organic acidurias, and fatty acid oxidation disorders (Rashed et al., 1997; Chace, Kalas, & Naylor, 2003; Chace, 2009); multi-analyte therapeutic drug monitoring (TDM) especially for the administration of cocktail therapies involving immunosuppressants (Constanzer, Chavez, & Matuszewski, 1995; Taylor et al., 1996; Yang, Peng, & Wang, 2005), oncology drugs (Lennard, 2001), anti-virals (Back et al., 2001; Gu & Soldin, 2007), etc.; toxicant and drugs-ofabuse screening where samples can be screened and validated in a single run (Sauvage et al., 2006b; Mauerer, 2007); the analysis of endogenous peptides especially where different isoforms exist; and the analysis of steroid hormones (Holst et al., 2007). With respect to the latter there has recently been a growing level of interest in the application of LC/MS/MS to clinical diagnosis in endocrinology to the point where the American Endocrine Society has issued a statement recommending LC/MS/MS for the determination of endogenous levels of steroid hormones such as

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testosterone over more traditional technologies such as immunoassays (Rosner et al., 2007). The rationale for this position has been the superiority of analytical results obtained by LC/MS/MS especially for low levels of these analytes (Gu, Chan, & Soldin, 2004; Holst et al., 2004). The reader is referred to a recent review article on the use of LC/MS/MS for a variety of endocrinology applications (Vogeser & Parhofer, 2007). Despite these useful applications and the rapid growth of LC/MS/MS in clinical diagnosis, the number of LC/MS/MS systems in use in routine diagnostic laboratories is actually relatively small compared to more traditional biochemical or immunological analyzers. For clinical diagnosis the major advantages of LC/MS/MS over traditional technologies include the speed of assay development, the same relatively low cost-per-assay whether there is one or many analytes in a single sample, high specicity, signicantly higher than immunoassays especially for small molecule analytes, and no costly analyte-specic reagents (ASRs) are required. There are also some disadvantages to LC/MS/MS: the high capital cost of these instruments usually with no reagentrental payment options; LC/MS/MS systems are complex instruments and it is not trivial to learn how to operate them; LC/ MS/MS is currently not sensitive enough for some important classes of analytes compared to immunoassays (e.g., proteins); and relatively labor-intensive or complicated sample preparation is sometimes required for trace analytes in biological matrices. Such sample preparation techniques are necessary because of the relatively high levels of proteins, salts, and lipids encountered in biological matrices which can interfere with the ionization of analytes should they co-elute. These disadvantages are responsible for the relatively limited uptake of LC/MS/MS into routine clinical diagnostic workows where the technicians who operate instruments are normally required only to place biological samples directly onto the analyzers with little or no pretreatment.

II. MULTI-DIMENSIONAL HPLC/MS/MS

How can MS be improved to achieve greater popularity and use in clinical diagnosis? An article just published in Clinical Chemistry (Annesley, 2009) interviewed ve clinical diagnostic MS users. The consensus was that, to make MS more routine in clinical diagnosis, sample pre-treatment, separation, and detection must become more integrated as they are in conventional clinical analyzers. To move closer to these goals many LC/MS/ MS users have recently been implementing on-line multidimensional chromatography to facilitate sample introduction, ease-of-use, and speed of analysis. Many of these are employing homebrew apparatus involving commercially available HPLC systems, multi-channel switching valves, a myriad of solidphase extraction (SPE) and analytical columns and, of course, mainly using triple-quadrupole analyzers. Many of these recent examples are for analytes with limited polarity such as steroids or non-polar lipids where the elimination of ion-suppressing interferences is essential for analysis. For example, isoprostanes (Haschke et al., 2007) are prostaglandin-like compounds formed in vivo from the free radical-catalyzed peroxidation of essential fatty acids (primarily arachidonic acid) without the direct action of cyclooxygenase (COX) enzyme. These eicosanoids possess potent biological activity as inammatory mediators that can affect the perception of pain. These compounds provide a specic and sensitive in vivo
Mass Spectrometry Reviews DOI 10.1002/mas

assessment of lipid peroxidation and as such are now widely used as an oxidative stress biomarker in both animal and human models. This research group used a homebrew 2-D LC/MS/MS system employing a C5 column to trap the isoprostanes from the protein-precipitated urine matrix followed by a C18 reverse phase (RP)-HPLC separation into a triple-quadrupole MS/MS instrument. Another diagnostically important but very challenging group of analytes are the thyroxines, specically the measurement of free thyroxine (FT4) and free triiodothyroxine (FT3) which are important for the diagnosis and monitoring of thyroid diseases (Thienpont, 2008). The concentrations of unbound or free forms of the thyroxines FT3 and FT4 in serum are extremely low and are also challenging to measure because they are not very polar. Serum samples must rst be dialyzed (Yue et al., 2008) or subjected to ultracentrifugation (Gu, Soldin, & Soldin, 2007) to allow the collection of free analyte without the protein-bound fraction. The group at ARUP (Yue et al., 2008) used dialysis and an on-line SPE LC/MS/MS assay that allowed a very rapid determination of FT3 and FT4. The dialyzed serum was injected directly onto a C5 guard column and washed and eluted onto the C18 analytical column. A very steep gradient eluted the analytes with good separation and narrow sharp peaks in <3 min. The lower limit of quantitation (LOQ) for FT4 was 1 ng/L and linear to >400 ng/L. Multi-steroid analysis is perhaps the most challenging diagnostic assay that benets greatly from multi-dimensional HPLC/MS/MS. A group from Leipzig (Ceglarek et al., 2009) has demonstrated the determination of a panel of 13 steroids in protein-precipitated serum using a 2-D system in which a POROS column is used for the on-line SPE and a monolithic RP-C18 column is used to rapidly separate, with high chromatographic resolution, the analytes of interest in a total run time of 4 min per sample. In this assay all the steroids elute within a 1.8 min time window with good chromatographic separation and narrow peak shapes. Comparison of the analytical results with those from immunoassays for some of the steroids was quite good except at the lower concentration ranges where it is well known that immunoassay performance is poor (Soldin & Soldin, 2009). LOQs were well into the ng/L range and recoveries and CVs were within acceptable ranges for most analytes. The use of steroid panels is of special interest for the diagnosis and treatment of complex endocrine disorders such as primary hyperaldosteronism, adrenal insufciency, congenital adrenal hyperplasia (CAH), Cushings syndrome, and disorders of gonadal function (Holst et al., 2007; Gu, Soldin, & Soldin, 2007; Soldin & Soldin, 2009). Among these endocrine applications perhaps the most important has been the use of adrenal steroid panels to conrm and monitor the treatment of newborns for CAH. Congenital adrenal hyperplasia refers to any of several autosomal recessive diseases resulting from mutations of genes for enzymes involved in the biochemical steps of production of cortisol from cholesterol by the adrenal glands. The incidence of this disease is approximately 1 in 15,000 newborns. Approximately 90% of cases of CAH are due to 21-hydroxylase deciency and 5% to 11-hydroxylase deciency. In most newborn screening labs dried blood spots (DBS) are screened for CAH by immunoassay for 17-hydroxy progesterone (17OHP), since elevated levels of 17-OHP are indicative of this condition. Filter paper or cellulose is an excellent sorbent to immobilize proteins and other similar potentially interfering
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matrix constituents. While the sensitivity of the immunoassay is adequate the specicity is rather poor as a screening modality, leading to relatively high false-positive rates (approximately 0.6%) (Janzen et al., 2007; Lacey et al., 2007). Recently, the Biochemical Genetics Laboratory (BGL) at the Mayo Clinic recognized that, by using a 12 min LC/MS/MS method to analyze the newborn bloodspot extracts monitoring a panel of these steroids including 17-OHP, adrostenedione (A), and cortisol (C), and using the ratio of concentrations (17-OHP A)/C, a much higher specicity could be achieved thus reducing the falsepositive rate by about an order of magnitude (Lacey et al., 2004; Marsden & Larson, 2004). Subsequently the group at the Hannover Newborn Screening Laboratory (Janzen et al., 2007) expanded the panel to include additional adrenal steroids in an attempt to increase sensitivity and specicity and highlight defects in other related enzymes such as 11-hydroxylase. Accordingly 21-deoxycortisol (21-DC) and 11-deoxycortisol (11-DC) were added to the existing panel of 17-OHP, A, and C, together with appropriate isotopically labeled internal standards (ISs) for quantication. They found that the ratio of concentrations (21-DC 17-OHP)/C correlated most signicantly with the CAH (21-hydroxylase) condition in newborns. Their analysis of the results on a cohort of 7,700 controls and 66 CAH positives indicated an increase in sensitivity and specicity to essentially 100%, a considerable improvement. Because the entire population is sampled in newborn screening, false-positive rates must be as low as possible to avoid needless and costly follow-up procedures. Considering that the true-positive rate for CAH is 1 in 15,000 births, even a ca. 1% false-positive rate, about what the current immunoassay exhibits, is much higher than it should be and places an enormous nancial burden on the public health system which would be required to locate and recall the babies for follow-up testing just to conrm a false-positive screening result. While the Hannover groups LC/MS/MS method provided a negligible false-positive rate, it still took approximately 6 min per sample making it unsuitable for newborn screening, but could be very useful as a second-tier conrmatory method (Schwarz et al., 2009). Janzen et al. (2007) speculated that if the analytical time frame could be shortened, the use of LC/MS/MS might be the best possible technology as a primary newborn screening modality for CAH. Subsequent to their earlier work described above, the group at Mayos BGL tried an on-line sample clean-up approach that could also be multiplexed. For many of these steroid panel assays most of the valuable MS/MS analyzer time is spent sampling LCcolumn efuent, where none of the desired analytes is actually emerging from the column. It is quite normal in LC/MS/MS analyses of trace endogenous analytes in complex matrices for signicant time to be spent waiting for the on-line SPE. Then more time is spent waiting for the matrix constituents to elute and for isobaric analytes to be separated, and still more time is wasted subsequent to the elution of the desired analytes for the late eluting matrix constituents to emerge from the column and for the column to re-equilibrate. The total run times can be as long as 10 or 12 min. Furthermore, the analytes of interest, being steroids of similar polarities, typically elute within a narrow 12 min window making the use of the expensive MS/MS analyzer only approximately 1020% efcient. The Mayo group (Lacey et al., 2006, 2007) employed an elegant solution to this problem by using a commercially available on-line sample preparation LC/ MS/MS system that was capable of multiplexing. (In this context
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multiplexing is the ability to analyze multiple HPLC efuent streams on a single MS/MS system without cross talk between HPLC channels.) Until very recently, in the world of commercially available multi-dimensional chromatography systems there have been only two options available, the Spark-Holland on-line SPE system (http://www.sparkholland.com/) and the TurboFlow system available from Cohesive Technologies (http:// www.cohesivetech.com/, now part of Thermo). (At the 2009 ASMS Meeting in Philadelphia, Applied Biosystems/MDS Analytical Technologies launched a dual-channel multiplexed LC/MS/MS system; however, very little information was available on the performance characteristics or capabilities of the system when this article was written.) Of the two currently available options only the TurboFlow system is capable of multiplexing (both sample preparation and LC introduction), allowing up to four channels to feed into one MS/MS instrument. Sample introduction is staggered in such a way that analytes emerge from each of the channels serially without overlap between channels. TurboFlow (short for turbulent ow chromatography) is an on-line SPE technology that allows proteins and ionic matrix constituents to ow-through to waste while analytes are retained for subsequent analysis by LC/MS/ MS. A diagram of a four-channel multiplexed multi-dimensional LC/MS/MS is provided in Figure 1. The principles of the technology are fully reviewed elsewhere (Wehr et al., 2000; Grant, Cameron, & Mackenzie-McMurter, 2002). Using this on-line clean-up and multiplexing technology the Mayo group (Lacey et al., 2006, 2007) analyzed an expanded panel of adrenal steroids similar to those of Janzen et al. (2007). The total run time including extraction was approximately 10 min per assay. However, because they used a four-channel multiplexed system the effective MS/MS analytical time per sample was really only ca. 2 min (see Fig. 2 for an example of multiplexed acquired data). Like the Janzen study, this work also demonstrated that expanding the panel of adrenal steroids to ve analytes increased the assays specicity and sensitivity, especially for the less-encountered CAH phenotype involving 11-hydroxylase deciency. Moreover, 2 min per newborn bloodspot is sufciently fast to open up the possibility of making LC/ MS/MS an economical primary screening tool for CAH, eliminating the need for the less specic 17-OHP immunoassay entirely. This could potentially save public health newborn screening programs considerable amounts of money through not having to deal with the expense of follow-up procedures or immunoassay systems and reagents. On-line sample clean-up and multiplexed sample introduction have both simplied sample handling and increased throughput considerably, aspects that are very important for the clinical diagnostic laboratory. Accordingly, these approaches have enjoyed signicant popularity recently. Large reference labs such as the Cleveland Clinic, Mayo Clinic, Quest, and LabCorp. have implemented these systems, usually for very high volume assays requiring the increased sensitivity and specicity of MS/ MS. For example, Clarke and colleagues at Quest have developed multiplexed on-line extraction methods for vitamin D (actually 25-hydroxy-vitamin D2 and 25-hydroxy-vitamin D3). Quest determines millions of vitamin D tests per year in this fashion (Clarke, 2008, personal communication), as well as estrogens in serum down to low pg/mL LOQs with throughputs of <2 min per sample (Redor-Goldman et al., 2007a,b), and high-throughput testosterone and dehydroepiandosterone (DHEA) assays (Goshal
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FIGURE 1. A schematic of a TLX-4 four-channel Turboow on-line sample preparation system. Each channel is composed of two switching valves that permit injected samples to be cleaned up on the Turboow column where they undergo de-salting, de-proteinization, and de-lipidation. After clean-up the sample is injected through the second switching valve onto the HPLC column where analytes are separated prior to MS/MS analysis. Multiplexing is achieved by staggering injections of analytes into the system. Before channel 1 is nished, the second sample is already injected into channel 2, and so on. The rst channel is ready for another sample prior to the nishing of channel 4. The results of such a multiplexed assay for ve enzymatic conversion products and their internal standards in conjunction with the determination of lysosomal storage disorders from newborn bloodspots are shown in Figure 2. [Color gure can be viewed in the online issue, which is available at wileyonlinelibrary.com]

et al., 2007; Clarke, 2008, personal communication). Similarly, Russell Grants group at LabCorp has utilized on-line turbulent ow sample preparation with multiplexed sample introduction for a wide variety of endogenous analytes including estrogens, vitamin D, free thyroxine, adrenal steroid panels, and other

steroids including testosterone and DHEA and cortisol (Grant et al., 2007; Patel et al., 2007). Ravinder Singh at Mayo has employed on-line sample preparation and multi-dimensional LC/ MS/MS for high-throughput assays for vitamin D (Taylor, Grebe, & Singh, 2005) and testosterone (Nelson, Grebe, & Singh, 2005),

FIGURE 2. The analysis of products of enzymatic conversion and their internal standards for the determination of lysosomal storage disorders (LSDs) is shown for four replicate calibration standards (lowest level standard injected four times). The analytes are coded by their respective enzyme (e.g., ASM, acid sphingomyelinase) and a full list of the analyte identities is provided in the original work (De Jesus et al., 2009a). Each assay takes approximately 4 min including on-line sample extraction. The HPLC run takes 3.5 min and the efuent is allowed into the MS/MS analyzer between 2 and 3 min from injection onto the HPLC column when the analytes of interest are emerging from the column. The injection of samples into the four-channel system (see Fig. 1) is staggered every minute so that the rst sample is injected into channel 1, the second a minute later into channel 2, and so on. Sample 5 is injected into channel 1 which, after 4 min, has had a chance to re-equilibrate, and so on until all samples in the queue are run. In this way, sample introduction is multiplexed and an analytical result is obtained every minute.

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and Sihe Wang of the Cleveland Clinic has employed turbulent ow on-line sample preparation and multiplexed LC/MS/MS to increase throughput and decrease costs on a vitamin D assay. Dr. Wang claims to have saved over 70% in reagent, personnel, and equipment costs in his rst year of operation using multiplexed LC/MS/MS with on-line sample preparation when compared to the same assay being run by radioimmunology assays (Wang, 2008, personal communication). It should be stressed at this point that many laboratories have successfully implemented their own homebrew multidimensional on-line sample preparation LC/MS/MS systems and have demonstrated impressive results. The missing dimension, however, is the increased efciencies and higher throughputs afforded by multiplexed sample introduction available on the commercially available system. Multiplexing is not trivial; a four-plex system comprises eight HPLC pumping systems, one for the clean-up column and another for the analytical column for each of the four channels. The software requirements and the ability to seamlessly interface to the mass spectrometers data system are also not trivial, and this is why home-built multiplexed on-line sample preparation LC/MS/MS systems are seldom if ever encountered in the literature. In addition to endocrinology, multi-dimensional chromatography with on-line sample preparation has made considerable inroads into the eld of clinical toxicology testing. LC/MS/MS has made a signicant impact on the TDM of therapies such as antidepressants and immunosuppressants, and is so widely used that there is even an FDA-approved kit of reagents available on the market for analysis of the immunosuppressant tacrolimus by LC/MS/MS (FDA-approved Waters Mass Trak Immunosuppressant Kit). On-line sample preparation enhances the ease-of-use of LC/MS/MS by permitting minimal sample-handling steps prior to introduction of a sample (usually serum or plasma) to the instrument. Marquet demonstrated the use of turbulent ow technology for rapid on-line sample clean-up followed by LC/ MS/MS for the simultaneous quantitative analysis of 20 analytes comprising 13 antidepressants and their major biologically active metabolites (Sauvage et al., 2006a). The total assay time was approximately 6 min, and all 20 analytes eluted within a 0.5 min window. Clearly this would have been a good candidate assay for multiplexing if greater throughput was required. Another example of on-line sample preparation is from the Leipzig group (Ceglarek et al., 2006) who employed the turbo-ow multidimensional system (TLX-1 from Thermo Scientic, San Jose, CA) but using a POROS column as the on-line clean-up medium. The unique aspect of this work was the ability to analyze ve immunosuppressant species (cyclosporine A, tacrolimus, sirolimus, and everolimus as well as mycophenolic acid (MPA) and its inactive phase II metabolite mycophenolic acid glucuronide (MPAG)) using the same method. Immunosuppressants are often prescribed as a cocktail therapy of more than one drug and each patient may require a different cocktail. By developing a universal method for the quantication of all possible immunosuppressants there is no need to stream the various samples since they can all be analyzed with the same assay method. What is also impressive about this assay is the fact that MPA can be accommodated since it is signicantly more acidic than the other species. Furthermore, the POROS on-line clean-up step actually separated the MPA from MPAG, a necessary requirement to avoid errors arising from in-source fragmentation of the MPAG ions contributing to the MS/MS signal for MPA.
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A Dutch group (Koster, Dijkers, & Uges, 2009) has demonstrated panel immunosuppressant detection in a single LC/MS/MS method using protein-precipitated whole blood. However, tacrolimus and cyclosporine A had to be measured separately from everolimus and sirolimus because of different de-salting protocols and MS/MS interface conditions between the two sets of analytes. Despite these peculiarities the throughput of the method was quite high at 20 samples per hour. More importantly these authors demonstrated that, for a sample load of 8,000 per year, the use of this LC/MS/MS method allowed them to save s110,000 per year in costs. Further efciencies and savings could of course be realized by multiplexing the assay with two channels. One of the most elegant examples of multiplexed on-line sample preparation LC/MS/MS is the high-throughput analysis of urine samples for fentanyl, a synthetic opioid with 80 times the potency of morphine (Crouch, 2008). Because of its high potency this prescription drug is increasingly tested in patients receiving it to ensure they are on their medication (and not selling it), and that they are not abusing the fentanyl by taking higher-than-prescribed doses. Some laboratories specializing in pain-management testing are required to run hundreds of urine samples per day. One such laboratory (Ameritox, Midland, TX) has implemented multiplexed on-line sample preparation LC/ MS/MS using turbulent ow chromatography involving four channels multiplexed into a single MS/MS system. Much of the therapeutic monitoring of analgesic drugs is currently being done using GC/MS which is the gold standard for such applications. Occasionally certain pain medications are better analyzed using liquid introduction techniques like LC/MS/MS because the drugs properties, such as thermolability or high polarity, make them incompatible with GC introduction. Alternatively they may be dosed at low levels making the higher sensitivity of LC/MS/ MS more suitable. The total LC/MS/MS run time for fentanyl was 4.3 min per channel. However, only the critical 1.3 min interval when the analytes of interest emerge from the HPLC is actually sampled by the MS/MS. By staggering injections from each of the four turbulent ow chromatography channels, the duty cycle of the MS/MS analyzer approached 100% efciency. Hundreds of samples can be run using a single MS/MS instrument on such a four-plex system each day (a sample injected every 1.5 min). At Ameritox, fentanyl was previously measured using GC/MS with a complicated sample preparation procedure. With the on-line extraction system sample prepara` tion was greatly simplied vis-a-vis the GC/MS method. Specically, no extraction or derivatization was required, and no evaporation or re-extraction steps were needed. A 15-step method for GC/MS was thus replaced by a simplied 5-step method for LC/MS/MS. In addition, tubes needed to be labeled only once for the turbulent ow workow compared to four times per sample for GC/MS, a potential source of human error. Thus, a four-plex on-line sample preparation LC/ MS/MS system was equivalent in output to four or ve separate GC/MS systems saving laboratory space, capital cost, and simplifying trouble shooting. It should be noted that the installations of high-throughput systems of this kind are fairly complicated despite their impressive records of throughput. What this means is that staff has to be suitably trained and the manufacturers sensitive to the support requirements especially during the early phases of installation and commissioning of such systems.
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III. MASS SPECTROMETRIC MEASUREMENT OF PROTEINS AND PEPTIDES IN CLINICAL DIAGNOSIS

Barr et al. (1996) demonstrated that a human disease biomarker protein could be quantied using isotope dilution LC/MS to monitor an ideotypic tryptic peptide, that is, a tryptic peptide used as a quantiable surrogate marker for the intact protein. Gerber et al. (2003) used stable isotope-labeled peptides and MS/MS to quantitate proteins and modied proteins by measuring their ideotypic tryptic peptides in a digest. Gygi coined the phrase AQUA peptides for Absolute QUAntitation when referring to the isotopically labeled peptides used as ISs for quantitation (Gerber et al., 2003). The advantage here is that the ideotypic tryptic peptide and its isotopically labeled IS AQUA peptide are chemically identical and therefore emerge from the HPLC column into the MS/MS for analysis at exactly the same time. This is ideal for quantitation, eliminating issues of poor ionization efciencies arising from ionization suppression due to co-eluting matrix components since both analyte and IS are ionized under the same conditions. The method selects precursor ionized molecules with the rst analyzer which are then fragmented and the appropriate product ions are monitored with the last analyzer (selected reaction monitoring (SRM)). The fragmentation reactions for peptides are dictated by their amino acid sequences and are therefore extremely specic. Quantication is realized by SRM using one or more fragmentations for the analyte peptide and taking the ratio of these signals to those of the same fragmentation reactions of the analogous isotopically labeled peptide IS (the so-called AQUA peptide). Leigh Anderson took this process a stage further with SISCAPA (Stable Isotope Standards and Capture by AntiPeptide Antibodies), utilizing immunoafnity to selectively concentrate the target peptides and thus increase the detection power of LC/MS/MS (Anderson et al., 2004). In this early work Anderson estimated that immunoafnity can increase the detectability of the ideotypic tryptic peptides and their isotopically labeled analogs by 23 orders of magnitude. The original embodiment of the apparatus was an on-line immunoafnity column which, after injection of plasma, could be automatically switched to release the peptides for quantitation by LC/MS/MS. Over the years this immunoafnity-LC/MS/MS scheme for protein quantitation evolved to incorporate use of magnetic beads coated with the immunoafnity reagents, implementing a trap washelute system that allowed for efcient transfer of the trapped ideotypic peptides to capillary LC columns for lowvolume high-sensitivity LC/MS/MS analysis. These new methods have demonstrated enrichments of target tryptic peptides of up to 20,000 times, making possible the detection of these surrogate tryptic peptides and the proteins they represent down to the low ng/mL levels thus rivaling some ELISA assays (Anderson et al., 2009). Certainly the ability to do multi-analyte protein determinations afforded by LC/MS/MS can be highly advantageous as multi-variate biomarker assays are discovered. Again the Anderson group has demonstrated the ability of LC/MS/MS to quantify tens of proteins in a single run with not much more trouble or expense than if a single protein was being measured (Anderson & Hunter, 2006). In this article they demonstrated the determination of over 100 SRM channels in a single LC/MS/MS run allowing quantitation of over 50 proteins.
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Hoofnagle et al. (2008) make a strong case for using LC/MS/ MS for certain protein biomarkers instead of using immunoassays, citing problems with potential interferences from endogenous immunoglobulins and imperfect concordance across platforms due to antigen microheterogeneity across patient populations. To illustrate these limitations they used peptide immunoafnity enrichment (SISCAPA) in concert with LC/MS/ MS to quantify thyroglobulin, a well-characterized tumor marker where interference from endogenous immunoglobulins affects results for 1025% of all patients. SISCAPA-LC/MS/ MS successfully detected tryptic peptides of thyroglobulin at picomolar concentrations (as low as 3 ng/mL) in non-depleted plasma while the trypsinization step also digested the endogenous immunoglobulins that potentially interfere with traditional immunoassays. This assay represents a step forward in overcoming some signicant limitations of the commercially available thyroglobulin immunoassays. Carr recently demonstrated (Kuhn et al., 2009) that SISCAPA-LC/MS/MS can be used to determine a panel of diagnostic biomarkers (two in this case) in patient samples taken at timed intervals during a planned myocardial infarction (PMI) initiated as a treatment for hypertrophic obstructive cardiomyopathy. In this proof-of-concept study a multi-analyte SISCAPALC/MS/MS assay was developed for two clinically relevant protein biomarkers of cardiovascular disease, cardiac troponin I (cTnI), and interleukin-33 (IL-33), the latter currently lacking a well-validated immunoassay. They did this to improve upon problems with the existing cTnI assay, such as potential proteolytic degradation of cTnI after sample collection, a problem that would likely not be a factor in SISCAPA-LC/MS/ MS. Furthermore, the authors speculate that multi-analyte (a.k.a. multi-parameter) analysis is theoretically more accurate with SISCAPA-LC/MS/MS than with immunoassays because there is likely less non-specic binding between the various antibodies (both capture and detection) and tryptic peptides versus endogenous proteins. Another potential advantage with SISCAPA-LC/MS/MS is that only a capture antibody need be developed instead of a well-matched pair of capture and detection antibodies, since the MS/MS is doing the detection. However, in the present example there was poor correlation between the cTnI SISCAPA-LC/MS/MS results and those derived by the immunoassay. One could speculate that, as with small molecule immunoassays, there could be non-specic interactions with endogenous antigens and the cTnI immunoassay to which SISCAPA-LC/MS/MS would not be susceptible. Unfortunately, IL-33 was below detectable levels in the patient samples, even though impressive detection limits were achieved by SISCAPALC/MS/MS (low ng/mL) using spiked serum. A recent study (Bondar et al., 2007) by a group at Mayo used LC/MS/MS on a more abundant putative biomarker for prostate cancer, Zn-a2 glycoprotein (ZAG), using the detection of an ideotypic tryptic peptide as a surrogate for the intact ZAG protein in serum. No abundant-protein depletion was required, and relatively high ow rate LC/MS/MS could be employed because of the relatively high endogenous levels of this putative biomarker. The method was validated between 0.32 and 10.2 mg/ mL, and the average concentrations for a small cohort of patients with prostate cancer and those for healthy controls were 7.6 and 3.7 mg/mL, respectively, a signicant difference. Despite these impressive early studies there is still signicant controversy as to whether the SISCAPA-LC/MS/MS
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assays, using ideotypic tryptic peptides as surrogates for endogenous protein biomarkers, will translate from the laboratories of highly skilled analysts into routine clinical diagnostic laboratories. This controversy was well captured in the recent article by Diamandis (2009) which comprised a Q&A session with experts in the use of these technologies. The consensus of opinion seemed to be that, while there is a lot of promise for this new technology, its greatest strength for the foreseeable future will lie in the validation of putative biomarkers, a function that cannot be undervalued. There has been over a decade of signicant research into proteomics which has led to a very impressive identication of approximately 5,000 proteins circulating in plasma. Despite these numbers the rate of newly FDA-approved immunoassay diagnostics per year has been in steady decline over this time until the number of new tests is between 0 and 2 most years. Recent articles have been decrying this abysmal record but there is now signicant hope that MSbased techniques like SISCAPA-LC/MS/MS will help in the validation process of this challenging backlog of newly discovered putative biomarkers (Carr & Anderson, 2008; Paulovich et al., 2008). Generally speaking, MS will likely be best used in situations where good immunoassays do not exist. One area where MS may also be very useful is for the determination of heterogeneous proteins whose isoforms respond similarly to the immunoassay but whose diagnostic values differ greatly. Nedelkov et al. (2005) used immunoafnity to capture and concentrate protein isoforms from human plasma samples, and subsequently used a mass spectrometer as a readout device. They observed a great deal of variation in a population of healthy volunteers when looking at point mutations and posttranslational modications of 25 endogenous proteins. The sources of variability could be genetic, environmental, or a mixture of both. This kind of variability in populations is precisely why large-scale multi-center clinical trials are performed on new diagnostic tests to ensure that this natural protein variability does not confound the interpretation of results. However, what is being ignored here is that the variability itself is likely a source of information about disease and possibly diagnostic of it. This was elegantly demonstrated in a subsequent publication by Nelson in which the various isoforms of B-type natriuretic peptide (BNP) were investigated in a cohort of 11 heart failure (HF) patients (Niederkoe et al., 2008). BNP is a well-established HF biomarker which has been incorporated into the guidelines for HF management (Nieminen et al., 2005; Swedberg et al., 2005). They developed an immunoafnity method (that they termed Mass Spectrometry Immunoassay or MSIA) that concentrates BNP isoforms prior to analysis by MALDI-TOF using a biotinylated BNP 32 as an IS for quantication. BNP is a 108 amino acid precursor that is proteolytically processed into BNP 32, the C-terminal 32-residue active form of the protein. Many HF patients testing for high levels of BNP paradoxically exhibit poor natriuretic response. The MSIA revealed that the plasma levels of the biologically active form of BNP, that is, BNP 32, were actually extremely low (ca. 30 pg/mL) even though the total immunoreactive BNP (iBNP) levels were measured to be at quite high levels (1,000 5,000 pg/mL) by a commercial point-of-care (POC) immunoassay kit. It is evident that the POC iBNP immunoassay kits exhibit cross reactivity between the inactive and biologically active forms of BNP, thus overestimating the natriuretic response. The actual low levels of BNP 32 measured by MSIA
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would indicate a natriuretic peptide deciency which could be alleviated by immediate therapeutic treatment with Nesiritide, a recombinant form of BNP 32.

IV. MEASUREMENT OF ABUNDANT PROTEIN BIOMARKERS

Here we return to applications of MS/MS to measurement of proteins by detection of ideotypic tryptic peptides in the presence of isotopically labeled standards. We will introduce several examples where the proteins of interest are not at trace levels as in the previous examples, but where nevertheless MS/MS provides some signicant advantages. The rst example involves the determination of human serum albumin (HSA) in urine as a biomarker for renal disease. Renal disease is on a steady increase given the complications associated with the widening epidemic of type-2 diabetes. The measurement of urinary albumin is a sensitive diagnostic and prognostic biomarker of renal disease. Disease prognosis is an increasingly important means of maintaining health especially in populations at risk such as those with diabetes. As discussed above, screening populations for a disease must be very accurate and precise to avoid both false negatives and false positives. They must also be inexpensive since many more tests will be performed to screen presymptomatic populations. A group at the Mayo clinic recently published an LC/MS/MS method (Babic et al., 2007) for the detection of urinary albumin using two ideotypic tryptic peptides and a novel way to generate the isotopically labeled peptide ISs. During sample preparation they introduced recombinant fully 15 N-labeled intact HSA as the IS. During tryptic digestion of the sample both labeled and unlabeled HSA are digested into tryptic peptides. This novel approach accounts for the possibility of less than quantitative HSA digestion, and is therefore superior to the usual method of simply adding isotopically labeled AQUA peptides after tryptic digestion. The LC/MS/MS method used was validated over the linear dynamic range of 3200 mg/L which spans the clinical requirements. Because HSA is present at sufciently high concentrations there was no need for preconcentration of the analyte and no need for nano-scale chromatography which is more difcult to implement in routine practice. Although the present example required long run times (30 min) it is likely this could be shortened and with multiplexed sample introduction assay times could be reduced further still. The LC/MS/MS method compared very favorably with immunoturbidity measurements but, unlike immunoturbidity, the LC/ MS/MS method did not require sample dilution to cover the full clinically relevant range. Another recent report utilized a similar LC/MS/MS technique to quantitate ceruloplasmin (CP) extracted from DBS for the newborn screening of Wilson disease (WD) (deWilde et al., 2008). WD is an autosomal recessive disorder of abnormal copper transport, resulting in markedly reduced CP in the blood. Decreased serum CP concentrations can therefore be used as an effective screen for newborns at risk of developing WD. WD has an estimated incidence of 1 in 30,000 individuals but is much more common in certain populations. WD is progressive and ultimately fatal if untreated, and relatively inexpensive effective treatments are available that prevent injury to the newborn if begun early. This disease is therefore an excellent candidate for newborn screening. The results of the study demonstrated clear differentiation between affected newborns and a carrier of WD
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based upon the analysis of newborn bloodspots. The levels of CP in a cohort of affected newborns ranged from 10 to 20 mg/L and for the unaffected carrier newborn the CP level was ca. 250 mg/L. The LC/MS/MS method was rather lengthy for a newborn screening modality (ca. 7 min). However, this could be made faster using a multiplexed multi-column system, as suggested by the authors. Another opportunity for MS/MS in newborn screening that appears to be extremely promising is the determination of hemoglobinopathies. These rare disorders are actually quite common in certain populations, and newborn screening permits early pre-symptomatic interventions that improve outcomes for babies affected by these diseases. Presently, screening for hemoglobinopathies occurs in every jurisdiction in the United States where techniques such as ion exchange (IEX) chromatography and isoelectric focusing (IEF) are implemented. IEX and IEF are somewhat time consuming and are prone to poor specicity, and therefore are not ideal as screening methods. There have been many articles published on the use of MS (particularly ESI-MS) for the determination of hemoglobin (Hb) variants through the molecular weight determinations of intact bHb chains (Rai et al., 2002; Bateman, Green, & Morris, 2008; Kleinert et al., 2008). These articles agree that ESI-MS cannot detect the zero mass change mutations (Lys $ Gln and Leu $ Ile). However, with careful measurements and sophisticated de-convolution algorithms, measurements of single mass unit differences are achievable but not easy to implement. Most laboratories make use of a combination of ESI-MS, IEX, and IEF to validate the identity of the mutation and ultimately are required to do genetic analysis (DNA sequencing) to conrm the disease. Grifths suggested that conrmation by MS/MS of tryptic peptides may be sufcient (Rai et al., 2002). Work by Dooley suggested (Awalt, Barnard, & Dooley, 2001) that essentially all hemoglobinopathies could be screened using MS/MS of trypsinized newborn bloodspot extracts, and be sufciently fast to permit this approach to be used by newborn screening laboratories. Recently, work by Dalton and Turner (Daniel et al., 2005, 2007) has elegantly demonstrated that, by using ideotypic tryptic peptides and those from wild-type globins as ISs, it was possible to screen for the clinically important Hb variants HbS, HbC, HbE, HbDPunjab, HbOArab, HbLepore, and bthalassemia as well as three other conditions of potential clinical signicance, namely delta b-thalassemia, hereditary persistence of fetal hemoglobin trait (HPFH) and alpha zero thalassemia trait, all in a single rapid experiment. Their methodology was simple: extract the newborn bloodspot, trypsinize the extract, and analyze by ow injection into the MS/MS instrument. The MS/MS method in most cases required only a single SRM channel for each Hb variant. The run times were short, between 1 and 2 min, making the technique suitable for newborn screening. This is possible only because Hb is the major protein constituent of blood (ca. 16%, w/v) requiring no depletion of other proteins, no clean-up, no pre-concentration, and no chromatographic separation. As suggested and demonstrated by Dalton and Turner et al., one added advantage of this approach was that SRM-triggered full scan MS/MS spectral acquisition provides amino acid sequence conrmation, thus obviating the need for costly and time-consuming genetic analysis follow-up. In other words, phenotype trumps genotype in hemoglobinopathy screening. This work was validated by a group in Belgium (Boemer et al., 2008) who repeated these experiments but employed at least two
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SRM channels per Hb variant. Using different brands of MS/MS instruments both laboratories demonstrated 100% sensitivity and 100% specicity for the detection of a wide variety of Hb variants as well as the ability to differentiate heterozygous genotypes. A recent clinical trial by the Turner and Dalton group (Daniel et al., 2008), using a cohort of 40,000 prospective newborn bloodspot samples, compared the predicate IEF technology with MS/MS. In the 40,000 newborn samples a variety of hemoglobinopathies was detected by both techniques. HbS was detected in 199 samples, of which 8 were HbS/HbF only and 3 HbSC. HbC was detected in 39 samples, HbDPunjab in 52, HbE in 48. No HbOArab or HbLepore mutations were detected by either method. There were no discrepancies between the two analytical techniques. The MS/MS had the added advantage that full scan product ion spectra could be used to obtain complete amino acid sequence information at the mutation site. This was demonstrated in a datadependent data acquisition mode where increased SRM signals automatically trigger the full scan data acquisition without perturbing or elongating the screening run. Another advantage of the MS/MS approach was that the consumables/reagent costs were <10% of those of the commercial IEF method. The MS/MS system used for the newborn screening of hemoglobinopathies is essentially the same as that currently used to determine >30 metabolic disorders by screening amino acids and acylcarnitines from newborn DBS, right down to the identical mobile phases being used for sample analysis. Based on this observation, the authors of this study suggested that the rationalization of technology platforms in newborn screening is now possible by consolidating screening for hemoglobinopathies and inherited metabolic diseases onto a common platform, MS/MS. One last example of the recent implementation of LC/MS/ MS into the newborn screening laboratory involves the indirect measurement of critical proteins. Certain diseases, for example, phenylketonuria (PKU), result from a congenital disorder that renders an enzyme dysfunctional. In the case of PKU the dysfunctional hepatic enzyme is phenylalanine hydroxylase, responsible for the conversion of Phe to Tyr. By measuring both substrate (Phe) and product (Tyr) from a newborn bloodspot by MS/MS and determining the ratio of concentrations, it is possible to detect PKU in the newborn with very high sensitivity and specicity, higher than measuring Phe alone (Chace et al., 1993). In PKU the substrate and product are readily measurable in bloodspot extracts. However, there are many diseases where the endogenous substrates and products are not readily measurable. One such set of congenital disorders that has recently attracted considerable interest are called the lysosomal storage disorders (LSDs). LSDs are caused by lysosomal dysfunction, usually as a consequence of deciency of a single enzyme required for the metabolism of lipids, glycoproteins, or mucopolysaccharides. Individually, LSDs occur with incidences of less than 1:100,000. However, as a group the incidence is approximately 1:5,000 1:10,000. Most of these disorders are autosomal recessively inherited but a few, such as Fabry disease and Hunter syndrome (MPS II), are X-linked recessively inherited (Winchester, Vellodi, & Young, 2000). The recent advent of viable enzyme replacement and bone marrow transplant therapies (Bruni et al., 2007), and the dire consequences of not treating affected newborns in a timely fashion, makes newborn screening for LSDs increasingly compelling. Several groups have been working on these assays, and Millington has summarized the history and clinical signicance of these tests in two recent
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editorials (Millington, 2005, 2008). By adapting standard methods in enzymology to newborn bloodspots and using uorescence detection, Chamoles demonstrated the determination of enzymatic activities associated with LSDs (Chamoles, Blanco, & Gaggioli, 2001a,b; Chamoles et al., 2002a,b). However, a signicant limitation of this technology was that it was not amenable to multi-parametric analysis (i.e., lacked the ability to monitor the activity of more than one enzyme per analysis). Quite independently a group at the University of Washington was developing an MS/MS-based method for the determination of enzymatic activity in cell lysates (Gerber et al., 2001). (An interesting footnote is that this approach using afnity purication and this particular kind of isotopically labeled IS was the predecessor to the ICAT reagents which played a transformational role in the quantitative analysis of whole proteomes.) The methodology exploits the use of a synthetic substrate which, in the presence of enzyme from cell lysates or extracted from newborn bloodspots, is converted into an easily detectable product. An isotopically labeled version of the product is used as the IS for quantication. After an appropriate incubation time and extraction of product and IS from the incubate, the extract is analyzed by ow injection into the MS/MS instrument using SRM. The ratio of responses for the product and IS is directly proportional to enzymatic activity. This group went on to demonstrate the analysis of galactocerebroside b-galactosidase activity directly from newborn DBS for the diagnosis of Krabbe disease using MS/MS (Li et al., 2004b). Chamoles and the University of Washington group subsequently collaborated to demonstrate the major advantage of the technique, which is that the analysis can be multi-parametric in that ve enzyme activities were measured in a single MS/MS experiment (Li et al., 2004a). Genzyme and the CDC have recently joined forces to make newborn screening of ve LSDs possible by supplying GMPproduced reagents and standards enabling government public health labs to more easily implement these assays (Dajnoki et al., 2008; Zhang et al., 2008; De Jesus et al., 2009a,b). The major motivating force is that these newly developed enzyme replacement therapies are only capable of saving affected newborns from injury if they are diagnosed as early as possible. This is truly one of the best examples of a theranostic test, that is, a diagnostic test required to determine whether a patient is a candidate for a specic therapy. One of the factors that may inhibit the acceptance of these tests is the signicant number of sample preparation steps. The current method requires liquidliquid extraction (LLE) followed by SPE. The cost and complexity of doing LLE and SPE on so many samples is not trivial. Despite these challenges, population screening for LSDs is proceeding in several jurisdictions. However, it is still early days for these screening programs and proper cost/benet analyses have yet to be done to fully justify the screening of these rare disorders. Fortunately, recent analytical developments at the University of Florence (la Marca et al., 2009) and the West Chester University of Pennsylvania (Kasper et al., in press) have suggested that on-line multi-dimensional HPLC sample preparation will obviate the need for the laborious and complicated manual sample clean-up procedures involved in the screening of LSDs. The approach of la Marca et al. used a homebuilt multidimensional LC in which the clean-up column was POROS and the analytical column C18. All analytes were separated in approximately 4 min. Herman and his collaborators (Kasper
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et al., in press) used a commercial TLX-4 multiplexed (fourchannel) system using a Turboow Cyclone column for on-line clean-up and a C8 column to separate the analytes that emerged from the column over a 1 min interval of a 3.5 min HPLC run. By multiplexing the assay with four channels Herman and his collaborators were able to decrease the sample assay time to 1 min, that is, a sample is injected every minute (see Fig. 2). For population screening, increasing sample throughput is as important as elimination of off-line and manual sample preparation steps.

V. METABOLOMICS AND DIAGNOSTIC BIOMARKERS

This review has described some of the current applications of MS in the eld of clinical diagnosis. Undoubtedly some important ones were overlooked. However, those that were covered were included because they exemplify the best attributes of mass spectrometric determinations compared to conventional diagnostic modalities, most notably the ability to look at multiple analytes in a single run providing unsurpassed sensitivity and specicity both analytically and clinically. One of the future drivers of the use of MS will be metabolomics. There has been a recent urry of interest in metabolomics which bodes very well for the discovery of new diagnostic biomarkers. Many of the putative biomarkers discovered by metabolomics are actually panels of endogenous metabolites, and MS has been demonstrated to be best suited for the analysis of ` biomarker panels vis-a-vis other diagnostic modalities. A recent review by Ellis et al. (2007) observed that of all the omics endeavors to discover new disease biomarkers, metabolomics is probably the most achievable. The number of expressible genes is $30,000 and the number of proteins expressed is likely close to 1,000,000 when one considers post-translational modications and splice variants. In contrast the number of metabolic products circulating in the human body is estimated to be <3,000, representing much less of a challenge to identify, quantify, and validate their diagnostic potential. Ellis review also discussed the motivation behind screening metabolic products as biomarkers of disease where early diagnosis leads to less morbidity and the less frequent use of costly interventions such as pharmaceuticals, hospitalization, and surgery (see Fig. 3). The earlier diagnosis of the inevitable onset of a disease such as diabetes, for example, would allow the patient to make healthier life-style and dietary choices before other interventions are necessary or morbidity occurs. There is a dedicated search underway for the discovery of prognostic markers that will enable the transformation of health management away from the treatment of illness toward the maintenance of wellness. Furthermore, these biomarkers will be of great practical value in patient stratication and monitoring to determine which patients are responding well to remedial treatments including pharmaceuticals. Like proteomics, however, the overwhelming majority of disease-related metabolomic studies, especially those studies using MS, have primarily involved the discovery of novel biomarkers and not their clinical or even pre-clinical validation. One group that has invested a great deal of forethought and resources in developing tools (i.e., kits) that can be used to economically perform rigorously quantitative metabolomic studies across relatively large sample sets is a company called Biocrates (pronounced like Socrates; www.biocrates.com).
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FIGURE 3. A depiction of the rationale (as explained in Ellis et al., 2007) that the best way to treat disease is to avoid it. The method of avoiding disease is to detect it early enough through the use of prognostic (i.e., presymptomatic) biomarkers before too much perhaps irreversible damage is done and before other costlier forms of therapeutic interventions are required. [Color gure can be viewed in the online issue, which is available at wileyonlinelibrary.com]

Such studies are vital for metabolomics research to be translated into clinical practice. One example of this application is a collaborative study by a group of systems biology researchers and Biocrates scientists to investigate potential biomarkers for diabetes by employing a mouse model (Altmaier et al., 2008). This work involved the use of a proprietary kit of reagents including all required reactants, standards, and protocols, analogous to microarray kits used in transcriptomics, but in this case used to determine over 800 endogenous metabolites by LC/MS/MS (SRM). A partial list of analytes is provided in Table 1 kindly supplied by Dr. Weinberger of Biocrates. Using these kits, murine blood plasma samples were analyzed from healthy and diabetic mice with and without experimental drug treatment. Concentrations of all the analytes were determined using isotopically labeled IS supplied with the kits. The ratios of the LC/MS/MS signals of analytes to those of respective ISs provided absolute concentrations that were used to calculate ratios of concentrations of the various metabolites. This large data set (>8002 640,000 data points across a signicant cohort of murine subjects) was subjected to sophisticated unsupervised bioinformatic analysis, which revealed new metabolic phenotypes of diabetes and the impact of medication upon them in a statistically objective manner. The study found that certain metabolites were oppositely impacted by the drug treatment, and that analyzing ratios between metabolite concentrations dramatically reduced the noise in the data set allowing for the discovery of new potential biomarkers of diabetes such as certain N-hydroxyacyloylsphingosyl phosphocholines. Using these statistical bioinformatics tools the authors were able to identify functionally related groups of metabolites indicating a diabetes-related shift, for example, from lysophosphatidylcholine to phosphatidylcholine levels. This rigorously quantitative methodology combined with bioinformatics data analysis can be readily applied to other drug testing scenarios and other medical disorders. A subsequent related article which further validates these rigorously quantitative methods was a publication from a
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collaboration between Biocrates and a number of Genomics and Systems Biology Institutes (Gieger et al., 2008). The authors represent this work as the rst genome-wide association (GWA) study with metabolic traits as phenotypic traits. In this work 363 metabolites in the serum of 284 male participants of the KORA study (Wichmann et al., 2005) were quantitatively measured by LC/MS/MS (SRM), simultaneously with the measurement of single nucleotide polymorphisms (SNPs) using genetic analysis techniques. Very clear genetically determined variants in the metabolic phenotype (the so-called metabotype) were observed. By using ratios of metabolite concentrations as a proxy for enzymatic activities, almost 30% of the genetic variability correlated with metabolic data with high statistical signicance (P-values of 1016 to 1022). The authors conclude that the investigation of the genetically determined metabotypes in their biochemical context may help to better understand the pathogenesis of common diseases and geneenvironment interactions. These ndings could result in a step toward personalized health care and early nutritional or other life-style interventions based on a combination of genotyping and metabolic characterization.

VI. ULTRA-HIGH RESOLUTION TECHNOLOGIES

What does the future hold for MS in the diagnostic laboratory? This writers prediction is nothing but double-digit yearly growth for the technology in this eld as new methods are developed and as it proves itself worthy of consideration both from a quality-ofdata perspective but also as a more cost-effective alternative to traditional clinical analyzers. The interesting thing about MS is that as a technology it is continually being improved and at a blindingly fast pace. For at least the last 15 years the sensitivity performance has increased by approximately an order of magnitude every 23 years, similar to Moores law applied to the efciency and speed of computers. Unfortunately this rapid pace of performance improvement cannot be sustained and the
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TABLE 1. A summary version of the Biocrates analyte table

Number of Compounds 41 Free Carnitine 21 19 proteinogenic amino acids Compound Type Acylcarnitines 40 Acylcarnitines with short, medium and long acyl residues Amino Acids 2 non-proteinoge nic amino acids (citrulline, ornithine) Biogenic Amines and Polyamines 14 ADMA, SDMA, Histamine, Methionine-sulfoxide; Kynurenine, Hydroxykyrurenine, Putrescine, Sp ermidine, Spermine, Serotonin, Phenylethylamine, Nitrotyros ine, Dopamine, Creatinine.

Glycerophospholipids 91 77 Phosphatidylcholines, 14 Lysophosphatidylcholines Sphingomyelins 15 10 Non-hydroxylated Sphingomyelins, 5 Hydroxylated Sphingomyelins Monosaccharides 1 Sum of Hexoses (including Glucose) Eicosanoids and other Oxidized 17 Polyunsaturated Fatty Acids

PGE2, PGD2, PGF2a, 8- iso -PGF2a, 6- keto -PGF1a, LTB4, LTD4, TXB2, 9S-HODE, 13S-HODE, 14(15)-EpETE, 12S-HETE, 15S-HETE, 15S-HpETE, 5S-HpETE, Arachidonic Acid, DHA Bile Acids

17

Cholic Acid, Lithocholic Acid, Cheno- and Ursodeoxycholic Acid, Deoxycholic Acid, 6 Glycine-conjugated bile acids, 6 Taurineconjugated bile acids Intermediates of Energy Metabolism

13

Lactate, Pyruvate and Oxaloace tate, PEP, Fumarate, Succinate, -KGA, Sugar phosphates, DHAP and 3-Phosphoglyceraldehyde, AMP, cAMP, 3-Phosphoglycerate Total and Free Fatty Acids

89

44 Fatty Acids (quantitative), 45 Fatty Acids (semi-quantitative) Oxysterols

16

7 Hydroxycholesterols, 3 Epoxycholesterols, Ketocholesterol, Dehydrocholesterol, Desmosterol, Cholestenone, Lanosterol, Dihydrolanosterol Phospholipids, Ceramides, Steroids, Vitamins : in development

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workhorse technology in this sector, the triple-quadrupole, is asymptotically reaching its maximum performance capabilities at least with respect to signal-to-noise and limits of detection. Recently new affordable benchtop MS platforms have been introduced that can routinely achieve resolving power of 100,000 and 12 ppm mass accuracies (Makarov, 2000; Bateman et al., 2009; Zhang et al., 2009). The signicance of these new technologies has yet to be fully appreciated. The specicity of such high resolving powers and mass accuracies may in fact rival those of the more commonly used triple-quadrupole MS/MS systems, and this would potentially have the effect of simplifying method development and analysis. With such a high degree of specicity in a single MS analyzer, analysts would not have to optimize two sets of instrumental parameters, ionization and MS/ MS. Furthermore, complex samples may only have to be run once under these conditions since even unexpected minor analytes will be measured with sufciently high specicity that, if these minor constituents were not originally targeted for detection, the data contained in the original data le would be sufcient to provide qualitative and perhaps semi-quantitative information upon reexamination of the original data le. One could readily envision all sorts of applications, especially in the study of complicated endogenous mixtures encountered in drug metabolism studies but especially in metabolomics experiments. One recent study that exemplies the use of this technology in metabolomics was recently published by Volmer who analyzed specically lipids extracted from plasma samples for a cohort of 10 healthy volunteers (Koulman et al., 2009). Approximately 240 separate data analyses were undertaken to develop a statistically meaningful data set obtained using the Orbitrap LC/MS system operated at 50,000 resolution, providing mass accuracies typically well below 2.5 ppm. UHPLC was used to provide maximum chromatographic separation capabilities with short run times. Like previous lipidomic studies (Schwudke et al., 2007) they found that setting m/z ion-extraction windows to 2.5 ppm provided unambiguous lipid identication based upon elemental composition. Moreover, the re-constructed ion chromatograms can be used for quantitation without signicant isobaric interferences using the analyzer constraints of 50,000 resolution and 12 ppm mass accuracy. No mention of the linear dynamic range was provided but the Orbitrap is specied to exhibit >4,000 linear dynamic range in a single mass spectrum (ThermoFisher Exactive, 2008). The large data set was subjected to unsupervised statistical analysis and demonstrated the ability to differentiate volunteers plasma samples and to also differentiate those samples enriched with a particular endogenous or exogenous analyte. The study demonstrated that, with high resolving powers and mass accuracies, both targeted and untargeted data analysis can be accomplished on the same data set. These results suggest that such a platform would be ideal for metabolomic studies, especially those involving human subjects where high numbers of analytes must be rigorously quantitated in a single run. However, such a platform must also be sufciently exible to be able to provide information on unexpected analytes such as exogenous compounds and their metabolites that may be present due to environmental factors. The translation of the Orbitrap from a research/validation tool to one used routinely for clinical diagnostic measurements is inevitable, given the analytical power of this novel technology and the results that have been published to date.
Mass Spectrometry Reviews DOI 10.1002/mas

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