Robert S. Plumb

It has been stated that dried blood spots (DBSs) represent the biggest step change in bioanalysis and drug metabolism and pharmacokinetics (DMPK) studies since the commercial application of liquid chromatography–tandem mass spectrometry (LC–MS-MS) in the late 1980s and early 1990s (1). This is a bold statement and one made born out of experience and foresight. The advent of the atmospheric pressure ionization interface in the 1980s (2) revolutionized bioanalysis and, indeed, most forms of pharmaceutical analysis. It allowed DMPK scientists to confirm analytes' presence, identify the structure of impurities and metabolites, reduce analysis times, and most importantly to bioanalysts, allow levels of sensitivities to be routinely achieved that could only be previously reached with complicated derivatization, gas chromatography–mass spectrometry (GC–MS) or electrochemical detection. During this era of LC–MS-MS analysis, we have seen assay sensitivities drop from nanograms per milliter to picograms per milliliter (3) and analysis time drop from 10–20 min to 1–2 min (4). There is no doubt that LC–MS-MS has revolutionized bioanalysis, so DBS has a tough act o follow if it really does represent the next big change in DMPK. 

DMPK analysis plays a key role in the drug discovery and development process. It provides high-quality preclinical analytical data to project teams and clinicians on the concentration and nature (parent or metabolite) of the drug-related material in a systemic system following dosing. This allows the pharmacokinetic parameters such as 

, AUC (area under the curve) bioavailability, and clearance to be determined. These are then used to calculate safety margin, dosing levels, and so forth. Traditional DMPK rodent safety assessment studies require relatively large volumes of blood, approximately 1 mL of plasma for each sample. To obtain a plasma sample in this amount requires taking approximately 1.5–2 mL of blood and sacrificing two to three laboratory animals for each time point on the pharmacokinetic curve.  

Widely used for screening newborns for metabolic disorders, dried blood spots have the potential to change how pharmacokinetic studies are conducted. The economic and ethical benefits of collecting blood for small rodent safety studies and clinical trials onto Guthrie-type filter paper cards (Figure 1) has been demonstrated by Spooner and colleagues (5–8) as well as others (9–11). The use of blood spot cards as a sampling medium requires only 15–20 µL of blood for each time point. This has several advantages: there is no need to sacrifice animals to obtain a sample; the pharmacokinetics curve produced is more representative as it is derived from one animal and not a composite; fewer animals are used; less compound is required; pharmacokinetic samples can be taken from the toxicological group rather than satellite groups; and there is no need to freeze samples and refrigerate them in transit. 

The use of DBS cards can translate into significant financial savings in terms of reduced animal use, limited or no requirement for frozen storage, and no need to ship the samples on dry ice. Indeed, in some geographies, the samples can be placed in a plastic food storage bag with desiccant, and placed in the normal post. This represents a significant cost saving. 

So What Does DBS Mean for the Bioanalyst and DMPK Biochemist? 

While the collection of DMPK samples in the DBS format can save a lot of money, it also represents a step change for animal technicians and bioanalysts. Animal technicians must learn a new way of collecting the samples, readily achieved via training videos. Bioanalysts must redevelop their workflows. Unlike working with plasma and serum where the samples are transferred easily by pipetting, diluted, and extracted with liquid–liquid or solid-phase techniques, DBS samples require a completely different approach. With DBS analysis, typically a 15-µL aliquot of blood is spotted onto the card in replicates of three for each sample from which a 3- or 6-mm punch is sampled, giving a sample volume in the region of 3–6 µL. The punching is carried out either using a robotic approach or manually with a handheld punch, which cuts out a clean circle of paper. The act of cutting out the sample as a small disk acts to give the analytical accuracy of the sample in the same way that pipetting a specific volume does with a liquid sample. The sample is then dissolved in a few hundred microliters of methanol, shaken for 1 h, and then centrifuged for 1 min. The supernatant is then removed and diluted with water (1:1) before injection onto the LC–MS-MS system.  

The blood spot cards are available in two main formats: treated and untreated. The treated cards are impregnated with chemicals to inactivate any bloodborne virus; however, the chemicals in the card that are dissolved during the extraction process can interfere with the analyte signal. It is therefore critical during method development to monitor the background signal from the card and  adjust the chromatography adjusted to properly resolve the card matrix from the analyte peak. This adds extra complexity as the analyst must address the challenge posed by the blood matrix and the new matrix components from the card. To illustrate, we analyzed a model pharmaceutical compound from three different dried blood spot cards. The samples, calibration line and QCs, were prepared by spiking an authentic standard of alprazolam in methanol solution into fresh rat blood. The samples were spotted with approximately 15 µL of fresh rat blood, onto a Whatman DMPK cards type A, B, and C. The cards were sampled using a 3-mm punch, dissolved in 100 µL of methanol, shaken for 1 h and then centrifuged for 1 min. The supernatant was removed and water added (1:1), before injection onto the LC–MS system. The chromatography was performed on a 50 mm × 2.1 mm ACQUITY BEH C18, 1.7-µm column (Waters Corp., Milford, Massachusetts), and the analytes were eluted under reversed-phase gradient conditions over 2 min using an ACQUITY UPLC system (Waters). The column effluent was monitored by electrospray MS operating in positive ion mode with the simultaneous collection of both multiple reaction monitoring (MRM) and full-scan MS data on a Xevo TQ mass spectrometer (Waters). The collision energy, capillary voltage, and cone voltage were optimized for each individual compound. The data displayed in Figure 2 show the results obtained. Here, we can see the untreated card (DMPK C) exhibited a significantly lower background signal than that of the treated cards. The full-scan MS data of each of the cards shows the treated cards have an increasingly intense ion current as the LC gradient increases. Notice, too, how the reduction in the signal response of the analyte ion with the treated cards compared to the untreated one (Figure 3). 

As we can see from this data, it is critical to the assay's success to develop the LC–MS methodology so that the analyte peak is clearly resolved from not only the endogenous material in the sample but from the background signal from the card as well. To achieve this, two technologies have been employed. The first of these technologies is sub-2-µm particle LC for maximizing separation performance and peak capacity, thus giving the analyst the opportunity to develop a robust method quickly. The second technology is a tandem quadrupole mass spectrometer that simultaneously acquires full-scan and MRM data, allowing the analyst to monitor the background signal while measuring the sensitivity of the analyte response. To achieve this performance, the MS system employed a novel collision cell, incorporating T-Wave design (Waters, Box 1), which allows the simultaneous collection of full-scan data and MRM data without loss in assay sensitivity. 

Bioanalytical assays are required to be reliable and reproducible. The approach to DBS bioanalysis was employed for the analysis of alprazolam, a highly potent short-acting drug of the benzodiazepine class. To evaluate the reproducibility of the blood spot assay. The analyte was spiked into blood to form a calibration line over the range of 100 pg/mL to 1000 ng/mL and spotted onto blood spot cards. The cards were dried for 3 h, a sample punched with a 4-mm punch, and the resulting sample dissolved in 200 µL of methanol. The samples were analyzed using an LC system coupled to a mass spectrometer. The separation was performed on a 50 mm × 2.1 mm BEH C

 1.7-µm column (ACQUITY, Waters), and the analytes were eluted under gradient conditions using a 5–9% methanol–aqueous ammonium hydroxide (0.1%). The resulting chromatogram obtained is shown in Figure 4 shows the extracted 100-pg/mL standard and blood blank as well as the resulting calibration curve.  

The data presented in Table I show the reproducibility of the QC data. We can see from these data that the low-concentration QCs had an RSD of 2.08% and the highest-concentration QC an RSD of 3.8%. This data shows that the high sensitivity assays can be developed with good reproducibility using blood spot cards and LC–MS-MS. 

Table I: Reproducibility of QC data from six replicas per concentration. 			

The detection, confirmation, and quantification of metabolites is a necessary part of modern bioanalysis for which MS is perfectly suited. For our work, the MS system collected MS and MRM data in the same analytical run to detect and confirm the identity of drug metabolites during a bioanalysis assay. The data in Figure 5 show the detection of the 4-hydoxy metabolite of alprazolam from a sample derived from a dried blood spot. The MS-MS spectrum acquired from the metabolite peak (Figure 5b) was triggered from the MRM signal related to the metabolite peak. The spectrum was obtained on the trailing edge of the peak using the enhanced sensitivity mode of ScanWave (Box 2), a data acquisition approach that increases the ability of scientists to confirm the identity of metabolite peaks during a bioanalysis assay.  

: The low sample spot and sampling approach means that only 3–6 µL of blood is used for the analysis. In a typical safety assessment study, the compound under test is dosed once or twice a day intravenously or orally at levels of 3–200 mg/kg. A drug compound or metabolite that is not sequestered into an organ or fat or rapidly eliminated typically circulates in the bloodstream at a level greater than 5 ng/mL, even at the later time points (24 h). These concentration levels are well within the detection range of today's modern LC–MS-MS analytical systems when operated in MRM mode. However, for low-dosed, rapidly cleared or inhaled compounds the circulating levels are typically much lower; in the 1–10 pg/mL range. Therefore, greater levels of sensitivity are required from the analytical instrumentation to accurately quantify these types of samples from a dried blood spot. This has forced many of the early adopters of DBS analysis to implement a two-tier strategy for sample collection, with sample collection for the orally dosed compounds based upon dried blood spots and the sample collection for low-exposure compounds employing the traditional serum–plasma approach. This results in the need for two different sample storage and preparation approaches, in some ways mitigating the cost-saving potential of DBS, as a provision must still be made for the storage and analysis of plasma-based samples.  

There are four major approaches to increasing sensitivity during a bioanalysis assay: Improve the sample clean-up; derivatize the sample; use a small scale of chromatography; and use a more sensitive mass spectrometer. Although the latter is the easiest and most popular option, it often can be used in conjunction with the first three items. Capillary and nano LC provide dramatically increased analyte response when the amount of material (injection volume) is small, just 1 or 2 µL (12). This increase in analyte response is due mainly to the fact that, when eluted from the column, the analyte is contained in a much smaller volume than with typical analytical or narrow-bore LC (12). There is evidence that superior detection levels due to greater ionization efficiency at low flow rates result from shifting to an electrospray ion source for the mass spectrometer (13). This only really occurs at single-digit nanoliter-per-minute flow rates, much lower than that encountered in capillary or nano-LC. While mainly employed for proteomics-type applications, there have been several papers describing the use of these small LC devices for small molecule type applications (14). In these applications, capillary or nano-LC was employed to analyze samples where the available volume is just a few microliters, for example tail-bled mice (15). To investigate the potential of this approach for blood spots, we investigated the use of a 300-µm column coupled to a tandem quadrupole mass spectrometer for the analysis of desmopressin, a synthetic replacement for vasopressin, the hormone that reduces urine production. The desmopressin sample was again spiked into fresh rat blood at levels ranging from 100 pg/mL to 100 ng/mL. A 20-µL sample droplet was spotted onto a DMPK A card. The cards were dried. Once dried,  a 6-mm spot was cut out and dissolved in 200 µL of methanol, shaken for 2 h, centrifuged, and an aliquot was removed for analysis. The DBS samples were analyzed using a 100 mm × 0.3 mm, 1.7-µm BEH C

 column (Waters). The analytes were then eluted with a reversed-phase acetonitrile aqueous gradient over 3 min at 12 µL/min. The column eluent was monitored in positive ion mode using the MRM transition 535→328. Desmopressin has a monoisotopic molecular mass of 1068.42 g/mol and one would expect the precursor ion to be MH+ = 1069, however the compound exists in several multiply charged states, +2, +3, and +4. Thus for this assay we chose to use the +2 charge state for the precursor ion and the product ion 

 = 328. The data collected was compared to that obtained from a 50 mm × 2.1 mm column (ACQUITY BEH C

, Waters) eluted with the same mobile phase and detected using the same MS-MS conditions. The data collected (Figure 6) show that the peptide was eluted with a retention time of 0.85 min on the analytical column and 1.6 min on the capillary-scale column. This difference in analysis time is due to the time taken to sweep out the delay volume in the capillary system. Although this volume is very low, at a flow rate of 12 µL/min there is still an observable offset between the two chromatograms. However, the dispersion on the capillary column is still sufficiently controlled to ensure that the peak width was just 3 s wide at the base. This allowed sufficient resolution of the analyte peak of interest from the endogenous components in the sample. Despite the slight delay in the separation, we can see that the method cycle time was still in the 3–4 min range; therefore, this form of capillary-scale chromatography could be used without deleteriously impacting sample throughput. The main aim of switching to the capillary-scale of separation is to improve assay sensitivity. The data in Figure 6 compare the signal response from the injection of the same amount of DBS sample onto the two column formats. We can see from this data that the capillary-scale system delivers approximately a 30-fold increase in sensitivity over the 2.1-mm narrow-bore LC. The theoretical increase in sensitivity is inversely proportional to the square of the column radii, in this case, 1.05

, a hypothetical increase of 49-fold. Though obtaining a 30-fold increase in sensitivity is in-line with theory, the difference can be attributed to the slight loss in peak width compared with the analytical scale LC. This 30-fold increase in sensitivity is a significant step forward in developing an analytical system that will allow DBS sampling to be used for more challenging low-exposure, low-dosed, high-clearance compounds such as inhaled products and biotherapeutics.  

The application of DBS sample collection holds the key to dramatic changes in DMPK studies. The ability to collect and analyze small sample volumes has the potential to reduce significantly the number of animals needed in safety assessment studies, improve patient recruitment in clinical trials, reduce costs, and improve data quality. The fact that the PK samples can be taken from the histopathology group and not a satellite group will ensure greater correlation of the data. Also, as the PK curve will be developed from samples taken from one animal rather than a composite of 2–3 per time point, poor absorbents and fast metabolizers will be detected more easily, whereas before they were masked by the sample-averaging approach. This approach does introduce a step change to the workflow of the bioanalyst and challenges them to develop new approaches to deal with the limited sample volumes. Technologies such as sub-2-µm LC and capillary-scale LC combined with data-rich MS are up to the challenge. If successfully applied, we could be witnessing the transition of DMPK from the LC–MS era to the blood spot age. And DBS could well be the biggest change in DMPK since the advent of electrospray LC–MS-MS in the late 1980s.  

The authors would like to thank Joanne Mather, Chet Bowen, and John Kehler of Waters Corporation for their contribution to the work discussed in this article. We would also like to thank Chris Benevides, Jim Murphy, and Mike Tomany with Waters Corporation R&D Department for their work on the design of the capillary nano-spray MS source. 

, Ph.D., is a visiting Professor at Imperial College, London, UK, in the Division of Surgery and Oncology. His role is the development of analytical technology specifically LC–MS to support the investigation in human metabolism using a metabonomics approach. He is the author of over 80 peer reviewed papers on the application of LC, LC–MS and LC–NMR-MS for metabolism studies. He is also a visiting Professor at Kings College London the Micro Separations Group with the Department of Pharmacy.       He obtained his BSc Hons from the University of Hertfordshire and PhD in Drug Metabolism from the University of Hertfordshire. Prior to his current role he worked for GlaxoWellcome in the Drug Metabolism Department for 14 years responsible for the identification of metabolites to support discovery and development DMPK using LC–NMR-MS. 

is a Principal Scientist currently working in the Pharmaceutical Business Development Group of Waters Corporation. He has over 10 years  experience in separation science with a strong emphasis in  chromatography coupled with mass spectrometry. 

, Ph.D., is a Section Manager of the Bioanalytical Sciences and Development group of the Worldwide Bioanalysis and Systems Management within PCD-DMPK at GlaxoSmithKline Pharmaceuticals R&D, based in King of Prussia, Pennsylvania. In this current role, he co-leads the world-wide effort responsible for the investigation, evaluation and implementation of novel technologies and processes to enhance the way that the quantitative bioanalysis of drugs is performed. One of the primary goals of this group is to continue the investigation into Dried Blood Spot (DBS) technology and fundamental. He obtained the B.S. in chemistry from the University of Maryland at College Park and his Ph.D. in Analytical Chemistry from Purdue University. Prior to his current role, Chris led a discovery-DMPK group for nearly seven years, also within GlaxoSmithKline.  

(1) N. Spooner, European Bioanalytical Forum, Barcelona, 2009, Oral Presentation.  

(6) N. Spooner, R. Lad, and M. Barfield, 

(7) M. Barfield, N. Spooner, R. Lad, S. Parry, and S. Fowles, 

(10) P. Bhatti, D. Kampa, B.H. Alexander, C. McClure D. Ringer, M.M. Doody, A.J. Sigurdson, 

(11) A.J. Wilhelm, J.C. den Burger, A. Chahbouni, R.M. Vos, and A.J. Sinjewel,

(12) K.R. Reid, C.F. Burant, and R.T. Kennedy, 

Articles

The collection of blood-derived samples from preclinical and clinical trial studies onto paper-based, Guthrie-type cards is gaining momentum within the pharmaceutical industry. This approach holds the potential to minimize animal usage, improve data quality, and reduce shipping costs. However, the small sample volumes and extra matrix effects from the cards result in method development and sensitivity challenges for bioanalysts. Here, we present a discussion on the analytical challenges that both liquid chromatography and mass spectrometry face as well as present some potential solutions to these issues.

Bioanalysis Using Dried Blood Spots: The Biggest Advancement in Bioanalysis Since LC–MS-MS?

Researchers and practitioners from various disciplines and subdisciplines within chemistry, biochemistry, and physics regularly depend on mass spectrometric analysis. Pharmaceutical industry workers involved in drug discovery and development rely upon the specificity, dynamic range, and sensitivity of mass spectrometry (MS). Particularly in drug discovery, where compound identification and purity from synthesis and early pharmacokinetics are determined, MS has proved indispensable. Biochemists expand the use of MS to protein, peptide, and oligonucleotide analysis. Using mass spectrometers, they monitor enzyme reactions, confirm amino acid sequences, and identify large proteins from databases that include samples derived from proteolytic fragments. They also monitor protein folding, carried out by means of hydrogen-deuterium exchange studies, and important protein-ligand complex formation under physiological conditions. Clinical chemists, too, are adopting MS, replacing the less-certain results of immunoassays for drug testing and neonatal screening. So are food safety and environmental researchers. They and their allied industrial counterparts have turned to MS for some of the same reasons: PAH and PCB analysis, water quality studies, and to measure pesticide residues in foods. Determining oil composition, a complex and costly prospect, fueled the development of some of the earliest mass spectrometers and continues to drive significant advances in the technology. Today, MS practitioners can choose among a range of ionization techniques that have become robust and trustworthy on a variety of instruments with demonstrated capabilities.

Two decades ago, MS was the preserve of experts and skilled technicians as the instrumentation required constant attention and adjustment. At that time, liquid chromatography (LC)–MS was in its infancy and atmospheric pressure ionization (API) source interfacing was just beginning. Samples requiring analysis were passed from the requesting scientist to these "experts for analysis." The samples would be analyzed, processed, and interpreted, and the results returned via a written report. Two decades later, the users and capabilities of LC–MS have changed significantly. Now mass spectrometers and LC–MS systems are ubiquitous in the analytical laboratory, especially in the pharmaceutical industry. These instruments are used by a wide variety of scientists for a diverse range of tasks, from purity screening in medicinal chemistry, to the quantification of drugs in blood and the identification of proteins for biomarker discovery. The usability of the current MS platforms has improved dramatically, with scientists able to operate the systems remotely via the internet, carry out complex data-dependent tasks, such as purification and peptide fragmentation to open-access systems where nonanalytical chemists can queue their samples for analysis and have the results e-mailed to them without ever having to know or concern themselves about the LC–MS process. 

 Figure 1: Daughter ion scans obtained with (top) and without (bottom) the ScanWave function. 

Recent reports put the number of LC–MS systems sold per year in excess of 2500 units. This large number of units sold each year is also reflected in the increased number of users. In 1980, the number of scientists attending ASMS was around 1250. By 2002, this number had risen to greater than 4000, with a growth rate of 10% per year. This growth in LC–MS users has occurred because of the increase in the number of samples analyzed each year per user, creating larger and larger amounts of high-quality data. More and more, this data is being turned directly into information or knowledge so that decisions are made in real time. Many of these new users have little interest in becoming expert mass spectroscopists and are instead looking for the instrumentation itself to decide the appropriate experiments to be performed as well as to interpret the data automatically and recommend a course of action (for example, pass/fail, pure/impure).  

Figure 2: Mass spectra of alprazolam and a plasma sample.  

The interfacing of LC with MS allowed analytical chemists access to about 80% of the chemical universe unreachable by gas chromatography (GC). It is also responsible for the phenomenal growth and interest in mass spectrometry in recent decades. A few individuals are singled out for coupling LC with MS. Beginning arguably in the 1970s, LC–MS as we know it today reached maturation in the early 1990s. Many of the devices and techniques we use today in practice are drawn directly from that time. 

In its simplest form, LC relies upon the ability to predict and reproduce with great precision competing interactions between analytes in solution (the mobile or condensed phase) being passed over a bed of packed particles (the stationary phase). Development of columns packed with a variety of functional moieties in recent years and the solvent delivery systems able to precisely deliver the mobile phase has enabled LC to become the analytical backbone for many industries. Continued advances in performance since then, including development of smaller particles and greater selectivity, also saw the meaning of the acronym change to high-performance liquid chromatography. In 2004, further advances in instrumentation and column technology achieved significant increases in resolution, speed, and sensitivity in liquid chromatography. Columns packed with smaller particles (1.7 μm) and instrumentation with specialized capabilities designed to deliver the mobile phase at pressures up to 15,000 psi (1000 bar) came to be known as ultrahigh-pressure liquid chromatography (UHPLC). Much of what is embodied in this current technology was predicted by investigators such as Prof. John Knox in the 1970s. 

Mass spectrometers can be smaller than a coin, or they can fill very large rooms. Although the various instrument types serve in vastly different applications, they nevertheless share certain operating fundamentals. The unit of measure has become the dalton (Da), displacing other terms such as amu. 1 Da = 1/12 of the mass of a single atom of the isotope of carbon-12 (

 C). Once employed strictly as qualitative devices — adjuncts in determining compound identity — mass spectrometers were once considered incapable of rigorous quantitation. But in more recent times, they have proved themselves as both qualitative and quantitative instruments. A mass spectrometer can measure the mass of a molecule only after it converts the molecule to a gas-phase ion. To do so, it imparts an electrical charge to molecules and converts the resultant flux of electrically charged ions into a proportional electrical current that a data system then reads. The data system converts the current to digital information, displaying it as a mass spectrum. 

The ions required in MS can be created in a number of ways suited to the target analyte in question. These methods include laser ablation of a compound dissolved in a matrix on a planar surface, such as by matrix-assisted laser desorption ionization (MALDI); by interaction with an energized particle or electron, such as in electron ionization (EI); or as part of the transport process itself, as we have come to know electrospray ionization (ESI), where the eluent from a liquid chromatograph receives a high voltage, resulting in ions from an aerosol. The ions are separated, detected, and measured according to their mass-to-charge ratios (

). Relative ion current (signal) is plotted versus 

, producing a mass spectrum. Small molecules typically exhibit only a single charge. The 

 is therefore some mass (m) over 1, with the "1" being a proton added in the ionization process (represented by M+H+ or M-H+ if formed by the loss of a proton), or if the ion is formed by loss of an electron, it is represented as the radical cation [M+.]. Larger molecules can capture charges in more than one location within their structure. Small peptides typically might have two charges (M+2H+), while very large molecules have numerous sites, allowing simple algorithms to deduce the mass of the ion represented in the spectrum. 

 includes the most notable technique, ESI, which itself provides the basis for various related techniques capable of creating ions at atmospheric pressure rather than in a vacuum. The sample is dissolved in a polar solvent (typically less volatile than that used with GC) and pumped through a stainless steel capillary, which carries between 500 and 4000 V. The liquid forms an aerosol as it exits the capillary at atmospheric pressure, and the desolvating droplets shed ions that flow into the mass spectrometer, induced by the combined effects of electrostatic attraction and vacuum. The mechanism by which potential transfers from the liquid to the analyte, creating ions, remains a topic of controversy. In 1968, Malcolm Dole first proposed the charge residue mechanism, in which he hypothesized that as a droplet evaporates, its charge remains unchanged. The droplet's surface tension, ultimately unable to oppose the repulsive forces from the imposed charge, explodes into many smaller droplets. These coulombic fissions occur until droplets containing a single analyte ion remain. As the solvent evaporates from the last droplet in the reduction series, a gas-phase ion forms. In 1976, Iribarne and Thomson proposed a different model, the ion evaporation mechanism, in which small droplets form by coulombic fission, similar to the way they form in Dole's model. It is possible that the two mechanisms might actually work in concert, with the charge residue mechanism dominant for masses higher than 3000 Da, and ion evaporation dominant for lower masses.  

The mass analyzer is the heart of the instrument and is a means of separating or differentiating introduced ions. Both positive and negative ions (as well as uncharged, neutral species) form in the ion source. However, only one polarity is recorded at a given moment. Modern instruments can switch polarities in milliseconds, yielding high-fidelity records. As well as separating the ions, modern mass spectrometers can trap and fragment ions (MS-MS or MS

 ) to produce a wealth of information about the molecule's structure. Other instruments, such as magnetic sector instruments, hybrid quadrupole–time of flight (Q-ToF), and ion cyclotron (ICR) mass spectrometers, can record the mass of a compound to 1 ppm, allowing for the elemental composition of a molecular ion or fragment ion to be deduced. The increased sensitivity afforded by modern MS over other forms of detection, such as UV and fluorescence, comes from the selectivity and specificity of the MS and MS-MS process. During these experiments, specific ions are allowed to pass through the analyzer and reach the detector. During a multiple reaction monitoring (MRM) MS-MS experiment, only ions that undergo a specific fragmentation are allowed to reach the detector. While this reduces the number of ions reaching the detector, it all but eliminates the noise, resulting in a superior signal-to-noise ratio (S/N). This improves assay sensitivity and specificity dramatically. The vast majority of quantitative experiments are performed on quadrupole-based instruments, whereas ion traps and accurate mass instruments are preferred for structural elucidation experiments.  

Single-quadrupole mass spectrometers require a clean matrix to avoid the interference of unwanted ions, and they exhibit very good sensitivity. Triple quadrupoles, or tandem mass spectrometers (MS-MS), add to a single-quadrupole instrument an additional quadrupole, which can act in various ways. One way is simply to separate and detect the ions of interest in a complex mixture by the ions' unique 

. Another way that an additional quadrupole proves useful is when used in conjunction with controlled fragmentation experiments. Such experiments involve colliding ions of interest with another molecule (typically a gas such as argon). In such an application, a precursor ion fragments into product ions, and the MS-MS instrument identifies the compound of interest by its unique constituent parts. 

An ion-trap instrument operates on principles similar to those of a quadrupole instrument. Unlike the quadrupole instrument, however, which filters streaming ions, both the ion-trap and more capable ICR instrument store ions in a three-dimensional space. Before saturation occurs, the trap or cyclotron allows selected ions to be ejected, according to their masses, for detection. A series of experiments can be performed within the confines of the trap, fragmenting an ion of interest to better define the precursor by its fragments. Dynamic range is sometimes limited in ion-trap instruments, and the finite volume or capacity for ions limits the instrument's range, especially for samples in complex matrices. 

Tandem quadrupole and ion-trap instruments have become the workhorses of modern analytical LC–MS(MS). The two capabilities have been incorporated into one instrument platform to produce a linear ion-trap instrument that had all the structural characterization benefits of ion-trap mass spectrometers with the quantitative capabilities of tandem MS instrumentation. These instruments have become popular with scientists who are required to perform more than one type of experiment (quantitative and qualitative) during the course of their work and require the flexibility to perform this on the same analytical platform. These tasks include impurity identification and quantification, and discovery DMPK, where both dosed parent concentration and metabolite characterization are required. Although they seem ideal, these instruments have some drawbacks, especially when using modern high-resolution chromatography. Here the chromatographic peak widths are so narrow (1–2 s) that there is not sufficient time for these ion trap mass spectrometers to select the ions for trapping, fragment the ions, and measure them to produce enough data points to define the peak accurately. Although the collection of MS-MS spectra can be performed with a standard tandem quadrupole MS instrument while still correctly defining the LC peak, the sensitivity is compromised due to the low duty cycle of the instrument.  

Along with this need to improve the usage and flexibility of the instrumentation is a requirement to improve the data processing and monitoring capability. The recent introduction of a new iteration of the tandem quadrupole mass spectrometer, using a traveling wave technology (as described by Kircher in U.S. patent 5,206,506), holds the potential to resolve many of these issues. The Waters Xevo TQ mass spectrometer employs a traveling wave technology to improve MS capabilities, allowing simultaneous multifunctional data acquisition such as MRM and product ion acquisition, all within a timescale compatible with sub-2 micron LC. The instrument is equipped with a modern tool-free source, which simplifies the process of routine maintenance and cleaning; workflow is also simplified with tuning, method generation wizards, as well as real time data checking functionality to prevent sample wastage if the analytical run fails for any reason. The instrument's software also features an interactive LC-MS method database that ensures the correct method parameters are selected and used. 

The mass spectrometer's T-Wave collision cell allows functionality such as rapid MRM switching, 20-ms positive ion–negative ion switching, and minimized cross-talk. This functionality makes the instrument ideal for rapid method development or use in a drug discovery environment for development of multicomponent assays.  

In these drug discovery environments, mass spectrometers often serve a dual purpose, as both a quantitative and a qualitative instrument in DMPK departments. The mass spectrometer provides the flexibility to perform both of these tasks in the same analytical run, or even the same eluted peak. This functionality is called ScanWave and is achieved by maximizing the duty cycle of the instrument. In conventional mass spectrometers, the selected ions enter from the first quadrupole (Q1) into the collision cell, where they are fragmented. The resulting fragmented ions exit the collision cell and are transferred through the third quadrupole (Q3) to the detector. This quadrupole (Q3) can act either as a selective filter, as in MRM mode, only allowing ions with a specific 

 value to pass through to the detector, or it can scan across the entire 

 value range, providing a full spectrum. This full scan mode is particularly useful when performing structural analysis; unfortunately, in this mode, conventional instrumentation suffers from poor duty cycle. This is because the ions exit the collision cell simultaneously regardless of their 

 value. However, as the third quadrupole scans, it can only measure or detect one 

 value at a time. Therefore, for a scan speed of 1000 

 per sec over a mass range of 1000 Da and a 2-s wide peak, the instrument will only spend 2 × 1/1000 s measuring each 

 value. This mass spectrometer's collision cell is designed to improve full scan sensitivity. In the last third of the collision cell, the fragmented ions are accumulated behind a DC barrier to effect ion enrichment. These ions are then released and contained between the DC barrier and an RF barrier at the end of the collision cell. The RF barrier is gradually reduced, ejecting the ions from the collision cell to the third resolving quadrupole. These ions are ejected according to their 

 ratio, with the heavier ions being ejected first. To improve the duty cycle of the instrumentation, the final quadrupole (Q3) is scanned in synchronization with the ejection of the ions from the collision cell, thus increasing the number of ions reaching the detector and hence the sensitivity. 

This increased scan sensitivity can be used to address several business and scientific needs in pharmaceutical analysis. The acquisition of this high duty cycle scan can be triggered during a standard MS experiment to provide structural information on the identity of an LC peak. In the field of bioanalysis, the functionality can be employed to confirm identity of a peak (product ion confirmation, or PIC). This is carried out within an MRM analysis. PIC works by taking one high-quality, high-sensitivity spectra after the apex of a peak and before the "touch-down" of a chromatographic peak. This does not affect the fidelity or accuracy of the peak quantification but allows for the acquisition of a product ion spectra to confirm the identity of the peak.  

In the disciplines of metabolite identification and impurity analysis, MS experiments such as constant neutral loss or common fragment ion analysis are often employed to detect ions that are related to the parent API molecule, or to look for particular metabolites that might be toxic, such as glutathione. Once the peaks of interested have been detected, a second analytical run is often required to obtain MS-MS structural information. The mass spectrometer can utilize its ability to perform either constant neutral loss or common fragment ion analysis and then rapidly switch to high-sensitivity MS-MS to obtain structural information. This capability removes the need for a second analytical run and hence improves productivity.  

Recent regulatory guidelines on bioanalysis have placed greater emphasis upon the measurement of ion suppression and control of the matrix and measurement of drug-related metabolites. The mass spectrometer's functionality facilitates the rapid switching between matrix molecules and analytes of interest. Phospholipids are a class of endogenous molecules that have been associated with ion suppression. These molecules can be monitored by measuring the parents of the common fragment ion, 

 = 184, which is associated with the choline polar head group. The MS system allows the simultaneous MRM monitoring of the compounds of interest and precursors of 

 = 184, allowing rapid, reliable method development. This capability also can be employed to monitor the background ions during the course of a clinical trial to evaluate any differences between patients due to phenotype, gender, age, or diet. This provides extra confidence in the results and allows anomalies to be explained. 

The usability and functionality of mass spectrometers has improved greatly over the last 15 years. Not only have they become more sensitive and capable of performing multiple experiments simultaneously, they have also become easier to use and have improved "up-time" and productivity. The advent of fast electronics and novel collision cell designs has allowed high-sensitivity full-scan MS-MS experiments to be performed at the same time as high-sensitivity MRM quantitative experiments. Concurrent with the increase in MS capability has been the move away from the expert MS scientist to the user tasked with answering a specific question, determining whether a product can be shipped, or monitoring food safety. The improved ease of use and intelligent software, which helps the general user achieve success in their task, will help push LC–MS even further into the general analytical community. 

The authors would like to thank Paul Rainville and Marian Twohig for their scientific contributions. 

 is Senior Applications Manager, Pharmaceutical Business Operations, Waters Corporation, Milford, Massachusetts.  

 is Principal Scientist, MS Technology Department, Waters Corporation, Milford, Massachusetts.

The authors discuss the changes that have taken place in the last two decades regarding the users and capabilities of LC–MS.

Robert S. Plumb

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