Cymoxanil (2-cyano-N-[(ethylamino) carbonyl]-2-(methoxyimino) acetamide) is the active ingredient in a number of pesticides manufactured by DuPont, for the control of various fungal diseases in crops and plants. It is mainly used for late blight potatoes, but is also applied to grapes, tomatoes, cucumbers and leafy vegetables. Its protection mechanism is to penetrate the surface to induce a host defence response to stop lesion growth and sporulation. 

The typical pesticide residue set at DuPont contains eight samples and usually takes an eight-hour workday to complete. The goal of this study is to take advantage of recent developments in sample extraction with a new laboratory grinder-mill and improved instrument sensitivity and selectivity using liquid chromatography tandem mass spectrometry (LC–MS–MS) to increase the number of samples analysed per day by a factor of three. The methodology conforms to the US Environmental Protection Agency (EPA) Office of Prevention, Pesticides and Toxic Substances (OPPTS) Residue Chemistry Test Guidelines — OPPTS 860.1340 Residue Analytical Methods.

  These were developed by the EPA as guidance for developing analytical methods to be submitted to the agency for review under federal regulations and dictate that average recoveries must be 70–120% with good reproducibility. 

Sample preparation for the analysis of pesticides and their residues has typically followed DuPont Report No. AMR 3705-95, "Analytical Method for the Determination of Famoxadone and Cymoxanil Residues in Various Matrices".

  In this procedure, ground-up samples are weighed into extraction jars, followed by the addition of water to allow them to rehydrate before extraction. Acetonitrile is added and samples are ground up with a laboratory homogenizer. The plant matrix is then allowed to settle-out and liquid extracts are filtered and collected in mixing cylinders containing sodium chloride. The mixing cylinders are capped, shaken and inverted to aid in the dissolution of sodium chloride and are then allowed to stand while the acetonitrile (upper layer) and water phases separate. 

Traditional Pesticide Analysis Using Liquid Chromatography

For the analysis of cymoxanil, the acetonitrile aliquot is back extracted with hexane, concentrated, diluted with water and passed through a conditioned strong anion exchange solid-phase extraction (SPE) cartridge stacked on top of a conditioned carbon black SPE cartridge. Cymoxanil passes through the strong anion exchange cartridge and is retained on the carbon black cartridge. It is then selectively eluted off the cartridge and the concentrated cymoxanil residues are dissolved in a hexane–ethyl acetate mixture. The resulting solutions are passed through silica SPE cartridges. Cymoxanil is retained, selectively eluted and concentrated into a methanol–water mixture adjusted to pH 3. Samples are filtered and analysed by high performance liquid chromatography (HPLC) with UV detection. 

Sample Preparation for Detection by LC–MS

There are approximately 20 clean-up steps in the AMR 3705-95 procedure, which limits the number of samples to 8–10 per day. A modification to this procedure, laid out in DuPont 13753, "Analytical Method for the Determination of Cymoxanil and its Metabolites in Leafy Vegetables Using LC–MS",

  significantly reduces the number of clean-up steps. In this procedure, samples are extracted using an acetonitrile–water mixture as described in DuPont Report No. AMR 3705 9, Revision 2. For cymoxanil, sodium chloride is added to an aliquot to separate the aqueous phase from the organic phase. The aqueous phase is discarded and the acetonitrile layer containing cymoxanil is passed through a strong anion exchange SPE column. The extract is then further cleaned up using a hexane liquid–liquid extraction followed by an Envi Carb (Supelco, Bellefonte, Pennsylvania, USA) SPE column. Cymoxanil is not retained on either of these columns, so the eluent is passed directly into an LC–MS system for analysis. Based upon the sensitivity and selectivity of LC–MS, the number of sample clean-up steps is reduced to approximately 10, allowing for the analysis of up to 16 samples per day. 

To further reduce the clean-up steps, a new sample preparation technique was evaluated that used a Geno/Grinder Model 2000 laboratory mill (Spex SamplePrep, Metuchen, New Jersey, USA). This is a laboratory mill or grinder specifically designed for vigorous vertical shaking of deep-well plates. It was originally designed to prepare plant tissue for extractions of nucleic acid, protein and other constituents by shaking the tissue, steel balls and a buffering agent together in each well of a titre plate. The application of this tool can be expanded to include micro-organisms when small silica beads are used instead of steel grinding balls. Microbes can also be disrupted in standard 96-well plates as opposed to deep well plates. Sample material that can be prepared includes bacteria, yeasts, molds, seeds, stems, roots, leaves and certain animal tissue. Because the vertical shaking motion of the equipment is so strong, many seeds and other forms of plant tissue can also be pulverized dry with the help of one or two grinding balls per well. 

For the leaves of watery crops such as tomatoes and potatoes, 10 g of sample was extracted twice with 20 mL of a 90:10 mixture of acetonitrile–water solution. To ensure complete extraction of the compounds from the plant material, steel balls were added to the samples to pulverize the plant material. The samples were then extracted by shaking at high speed at approximately 700–1200 cycles/min, depending upon the sample. The sample extract was brought to 50 mL by adding acetonitrile. Approximately 1 mL of the extract was filtered using strong anion exchange and the SPE process described earlier. A 100 µL volume of sample was diluted to 1.0 mL in an HPLC vial with mobile phase and was analysed directly by LC–MS–MS. This approach cuts down the number of clean-up steps to three compared to 20 with the AMR 3705-95 method and 10 with the DuPont 13753 method. 

To evaluate the efficiency of the sample preparation and clean-up stage described previously, cymoxanil and its various metabolites were determined in a number of wet and dry crop and plant materials. All samples generated were analysed using LC–MS–MS. 

This instrument is coupled with a 1100 Series HPLC system (Agilent Technologies, Santa Clara, California, USA) to first separate the biological species of interest, based upon their elution times off a column. The eluent is then introduced into the mass spectrometer for ionization to identify and quantify the species using triple-quadrupole MS. The principle of this technique is based upon confirmation of a particular molecular ion by the generation of its product species using collisionally induced dissociation or fragmentation.

  This technique, commonly known as multiple reaction monitoring (MRM), uses two resolving quadrupole mass filters, separated by another quadrupole, which is slightly pressurized by the introduction of a collision gas such as hydrogen or helium. In this MS–MS mode, the first quadrupole is used in a mass-resolving mode to select the precursor ion. The second quadrupole (or pressurized multipole collision cell) is used to produce fragmentation of the precursor ion. The final quadrupole is used in a mass-resolving mode to provide mass analysis of the resulting fragmented product ions. These species are then compared with reference spectra-data to produce unambiguous identification of the biomolecules of interest. 

 Table 1: Brief summary of the HPLC and LCâMSâMS instrumental conditions used for this study.			

The benefit of the MS–MS design is that it also can be used in MS mode for quantification purposes. In this mode, the first quadrupole is typically used in the rf-only mode, as a wide bandpass filter to transmit molecular ions of a wide mass range. The collision cell is also used in the rf-only mode, but this time no collision gas is flowing, so the cell is just used to transmit ions to the last quadrupole, which is used in the mass resolving mode. This is known as the selective ion monitoring (SIM) mode. The ability to switch between both modes maximizes the amount of data being generated, particularly when small amounts of sample are being analysed. The higher number of product or fragmented ions identified leads to better confirmation of the parent molecules. This is especially important when analysing data from an LC separation. Cymoxanil, for example, which is an aliphatic nitrogen compound with a formula of C

 and molecular weight of 198, is typically eluted from the column after 10–15 min, over a period of 20–30 s. For this reason, it is very important to be able to switch very rapidly between SIM and MRM mode to collect both the molecular and fragmented information for positive and unambiguous identification of these species. After identification is made in the MS–MS mode, quantification of unknown samples can be performed in either the SIM or MRM modes. 

Reversed-phase LC was used to separate cymoxanil and its metabolites from the rest of the extractants. The LC–MS–MS instrument was operated in single-ion monitoring MS negative-ion mode for quantitative analysis. Peak area was used for quantification. For confirmation of the presence of the analyte in unknown samples, the relative intensities of the fragmented ions were measured using the MRM mode. A brief summary of the HPLC and LC–MS–MS conditions are shown in Table 1. 

A 0.01  ppm cymoxanil LOQ fortification sample (following method DuPont 13753) and control (blank) in the SIM mode is shown in Figure 1. It can be seen very clearly that there is a significant peak eluted at about 15 min, which corresponds to the cymoxanil. 

Confirmation of the compound is then made by measuring the intensities of the fragmented product ions in the MS–MS mode, by comparing them to known ratios in a database of pesticides. The mass spectrum (60–300 Da) of cymoxanil in single MS mode is shown in Figure 2, and the resulting calibration curve for 0.2, 0.5, 1.0, 5.0 and 10.0 µg/L in SIM mode (

Table 2: Percentage recovery data and RSD for a 0.05 ppm (LOQ) and 0.50 ppm (10 x LOQ) reference samples run using three different analytical methods.			

Table 2 shows some typical recovery data and RSD for 0.05 ppm and 0.50 ppm reference samples run using three different analytical methods. 

Sample Preparation for Field-incurred Residues

Once the methodology was optimized for the separation and quantification of cymoxanil, the laboratory mill sample preparation and clean-up procedures were used for comparison purposes with traditional sample preparation methods. The basis of this method was then used to investigate an experimental new insecticide (DuP-1) and to identify and quantify its active ingredient and metabolites in various wet and dry plant and crop materials with field-incurred residues. Rather than fortifying directly onto a control sample in the laboratory, the crops were treated in the field with the insecticide at the timing and rate of typical agricultural practices. The crops were harvested at maturity and all detected metabolites have been incorporated into the plant matrix rather than on the surface, as is the case for laboratory fortifications. The results of this experiment will further test the extraction efficiency of the laboratory mill sample preparation procedure, which is shown in the following: 					

Extract twice with acetonitrile–water in the grinder for 2 min at 1200 cycles/min.

Dilute 1 mL of extract to 5 mL with water (adjust to pH 2.5).

Filter through strong anion exchange SPE and collect the extract.

Filter through a PTFE membrane into an LC vial.

The total ion chromatogram (TIC) generated by the LC–MS–MS in MRM mode of the new experimental insecticide under investigation and its metabolites (A–G) is shown in Figure 4. Two pairs of metabolites — A and G, as well as D and E, are eluted from the LC column at the same time, indicating that they have similar chemical properties. A benefit of the LC–MS–MS method is that by monitoring the individual transitions of the MRM for each metabolite, the overlapping peaks can be resolved. Metabolite D with a mass of 444 Da can be identified by monitoring the transition 443 (molecular weight  1) to 298 Da and separated from metabolite E, which has a mass of 458 Da and a transition of 457 to 188 Da. Figure 5 shows the resulting MRM chromatograms for a 0.2 ppm standard of the metabolites D and E (top), together with a tomato sample spiked at the LOQ (bottom) and illustrates good resolution of metabolite D from E. 

Table 3 compares the new sample preparation and clean-up method with a traditional homogenizer probe (Tekmar Tissumizer, Cincinnati, Ohio, USA) sample preparation using the DuPont-13753 method described earlier. It shows quantitative data (parts per million in sample) for the experimental new compound and one of its metabolites (D), for various crop samples. This methodology was then used to analyse a variety of wet, dry, oily and acidic crops for the active ingredient together with its metabolites. Table 4 shows spike recovery and RSD data for one of the watery crops (tomato), showing that the DuP-1 insecticide and all its metabolites are within the guidelines stated in EPA OPPTS 860.1340, which states that test data must have average recoveries of 70–120%. Although they are not presented here, the study also looked at other crops including limes (acidic crop), almonds (oily crop) and wheat straw (dry crop) and achieved similar spike recoveries and precision values. 

Table 3: Comparison between the Geno/Grinder and Dupont-13753 sample prep methods for the quantitation of the experimental new compound (DuP-1) and one of its metabolites (D) in a variety of wet and dry crop samples.			

It has been shown that the extraction of pesticide residues using a novel sample preparation procedure and identification and quantification of its active ingredients and metabolites using LC–MS–MS provides an extremely efficient, rugged and high-throughput analytical method. Compared with the traditional sample preparation, clean-up and detection methods, sample throughput was increased by a factor of three, from 8 samples to 24 samples per day. It also demonstrates that the extraction efficiency of the new procedure is valid for field-incurred residues in addition to laboratory-fortified samples. 

Pesticides and Toxic Substances (OPPTS) Residue Chemistry Test Guideline OPPTS 860.1340, Residue Analytical Methods

Articles

Regular article

The high-throughput analysis of pesticides in raw agricultural commodities has been hindered by the slow and laborious sample preparation stages. Using traditional clean-up procedures, as many as 20 steps are required to get the sample in a convenient form for analysis, which typically limits sample throughput to eight samples per day. This study evaluates a new approach using a novel laboratory mill and pulverizer to extract various pesticide residues from different kinds of plant materials and to identify and quantify the active ingredients by liquid chromatography tandem mass spectrometry (LC–MS–MS). It shows that by automating the procedure, the number of clean-up steps can be reduced significantly, which increases sample throughput over traditional approaches.

A Novel Sample Preperation Approach to Increase the Throughput of Pesticide Analysis by LC–MS–MS

In 1997, in an article that appeared in what then was 

), I departed from my usual technical writing, which up to then had been primarily for peer review. The 

 editor had requested help recognizing the publication's 10th anniversary. Mass spectrometry (MS) and liquid chromatography–mass spectrometry (LC–MS) in particular, given the rapidly evolving commercial landscape, I thought made investigating the phenomenon worthwhile. Little did I realize how much effort a writer must devote to developing and presenting ideas in finished form. I must have forgotten that lesson when, five years later, I agreed to write the "MS — The Practical Art" regularly. 

Nearly 35 bimonthly columns later, I happened across that initial article, "Commercialization of LC–MS 1987–1997: A Successful Decade in Review" [

(11), 728–737 (1997)]. It made me curious about just how many technological advances the field of MS had witnessed during the ensuing decade and what had not changed. 

Of course, the technological basis, or history, remains unchanged for what was then called the "hyphenated" on-line practice of LC–MS. What has changed, though, is this: as an analytical method, LC–MS has become so ubiquitous in industry and research that more practitioners now approach it as a single, integrated analytical process. Based upon the rapid growth of the American Society of Mass Spectrometry (ASMS) indicated by the annual conference attendance in recent years, the cross-section of who constitutes today's practitioners has broadened as well. We have abandoned the sense of technological duality that the hyphenated usage implied. My 1997 article attempted to outline evolution of the mass spectrometer as it was becoming a true mass detector "mixing significant technological events with significant commercialization initiatives over [those] 10 years." It fleetingly acknowledged important events in the development of LC–MS to that point, such as Blakely and Vestal's 1983 publication on thermospray which, though no longer used, was the first LC–MS enabling technology.

John Fenn (along with Koichi Tanaka and Kurt Wüthrich) received the Nobel Prize in Chemistry, in 2002, for his work on the most significant aspect of LC–MS practice — electrospray ionization (ESI) — based upon work published in the early 1980s.

  Among those receiving credit for commercializing the concept were Bruins and Covey who, along with Henion, published commercially promising ESI designs in 1987.

A harbinger of things to come, the first truly hyphenated system appeared in 1982. Hewlett Packard's mass spectrometry detector (MSD) used a gas chromatograph to introduce analytes into the mass spectrometer's ion source. Of course, the gas chromatograph limited the MSD to analysing only compounds of sufficient vapour pressure. However, no such restriction attended coupling a high performance liquid chromatography (HPLC) system to the mass spectrometer. LC–MS had its own problems: vacuum system and flow-rate requirements evolved large, costly, often unreliable instruments. 

In 1993, Waters (Milford, Massachusetts, USA) introduced a benchtop instrument for LC–MS. Designed to operate using particle beam technology, which in the era of ESI exhibited certain performance issues, it nonetheless deserves footnote-level respect for giving us true electron ionization (EI) spectra for an LC separation. That said, the first LC–MS designed for widespread adoption is probably Vestec's thermospray-equipped, modified HP MSD, which the company offered at the 1986 Pittsburgh Conference and Exposition (Pittcon) for the almost magical price of $100000. Thermospray of course faded quickly and so did the dream of vastly capable but inexpensive LC–MS. 

As is so often the case, the optimism of commercial interests proved unwarranted. In 1988, a year after Extrel Corporation had introduced particle beam technology at Pittcon; Hewlett Packard (now Agilent Technologies, Santa Clara, California, USA) introduced a version derived from the original particle beam instrument. The HP product manager claimed the interfacing of LC and MS brought to the LC–MS market the reproducibility of results and ease-of-use characteristic of gas chromatography (GC)–MS. In those days, industry watchers estimated market sales to be $15 to $20 million, with thermospray instruments accounting for 60–80% of the business.

  Always a point of industry contention and one whose public discussion is typically avoided for reasons of competition, Finnigan (now Thermo Fisher Scientific, Waltham, Massachusetts, USA) management succinctly stated at Pittcon 1993 that estimates of market size were almost "guaranteed" to be incorrect and were "probably rather conservative". Indeed, the total market for 1993 was predicted at $640 million. In the years immediately following, it became obvious that both sides of the argument were incorrect.  

The early 1990s fell far short of sales expectations, the consequence of a general economic slowdown. (Remember, these were the days when costly analytical purchases were driven largely by a rapidly building pharmaceutical industry.) A 1996 American Chemical Society report cited 1991 sales exceeding $450 million, but with the caveat that "growth in these areas is difficult to predict." 

Between 1985 and 2004, it was my good fortune to co-organize the LC–MS interest workshop at the annual conference of ASMS (

). During that time, the success of LC–MS transformed ASMS. Once an annual event for thought leaders and innovators in MS research, the conference also came to represent an educational resource for nonspecialists. In 1987, 2200 members attended. By 1997, that number had doubled. In keeping with the growing interest, attendance at the LC–MS interest workshop grew, by some estimates to more than 1200 from a few hundred. 

I began taking note of changes in abstract topics matched to overall attendance during a stint on the programme committee in 2003–2005 (and in the years since — the conference administrator, Brent Watson, kindly obliges with statistics each year). The ASMS conference has grown in the early years following 2000 to nearly 6000 attendees. Once the LC–MS interest wave crested and LC–MS became the ubiquitous tool it is today, the conference organization reflected that interest. In these much larger conferences, LC–MS is no longer the esoteric focus of a special interest group. Indeed, the technology now appears as a topic or reference in virtually every discussion. As for the LC–MS interest group, it has reverted to a more reasonable size, returning to a more utilitarian, narrow charter. 

In the early years, presentations rarely associated LC columns and separations with LC–MS practice. LC served merely as an "inlet" for compounds dissolved in the condensed phase, and it was the mass spectrometer that bore the brunt of separations work. A survey in 1985 noted that of 400 chemists interviewed, less than one third were "comfortable with prospective applications and operation of LC–MS".

  showing comparative analyte ranges for the major LC–MS interfaces appears quite dated today: particle beam and thermospray, two of the four noted along with atmospheric pressure chemical ionization (APCI) and ESI, pretty much vanished years ago. On the other hand, I noted in my article emerging technologies just gaining ground:  

"Significant recent introductions, with much shorter commercialization histories (as of this date), such as the reengineered ion-trap (commercialized initially by Finnigan, now Thermo Fisher Scientific) and time-of-flight (TOF) instruments also are worth a passing note for their potential role in shaping the future direction for LC–MS detection."

It was obvious by the time the article appeared that a few years earlier, "The interface with the greatest potential impact was... previewed as a commercial possibility in 1983,

  when preformed ions in solution were described... The advantage of electrospray, readily embraced by protein chemists, was the ability to charge multiple sites on... relatively large molecular weights... well above the operating range of the quadrupole." 

Looking back over the 16 years since the article was published, it is obvious that quadrupoles still serve as the analytical backbone of LC–MS practice. Evolution of the technology brought ion traps to prominence from their attendant problems of space-charge effects (too many ions in the trap) and quantification issues to much-improved, linear traps coupled with ion cyclotron devices such as the Orbitrap (Thermo Fisher Scientific) or as an adjunct to tandem designs (MDS Analytical Technologies Sciex, Toronto, Ontario, Canada). 

Consider that in recent years, the ASMS conference attracts about 6000 attendees who submit almost 3000 abstracts for talks and posters (low of 2730, in 2009, and recent high of 2907, in 2008). Granted, we must bear in mind the content in recent years has become heavily weighted away from environmental and industrial applications toward life science (searching for individual instances where an abstract contains the term "peptide" for instance returns 1023 hits out of 2730 abstracts), but the consistency in attendance numbers year-to-year can reveal some shifts in technology adoption trends. For instance, "ion trap" and "quadrupole" are too common (500 or more instances for each across numerous applications). Moreover, the ASMS programme committee rules out trade names as part of the acceptance process. Yet some such terms are unavoidable and unique. "Orbitrap", though a trade name, is an example of commercialized novel technology. Since 2006, Orbitrap cites per abstract rose from 39 at its introduction to ASMS to 429 in 2009. Similarly, we can judge unique entities such as SYNAPT (Waters Corporation) and the ability to combine ion mobility as an orthogonal discriminator (in essence multiplying the resolution or peak capacity of a mass spectrometer), which shows a tripling of its unique instances since 2007. 

The ASMS conference is, of course, an MS venue. With that in mind cites for "UPLC" (Waters' ultra high performance LC system) more than tripled, to 142, since 2006, which is impressive because LC is simply an "inlet" device to a mass spectrometer even today (by comparison, "HPLC", long-established as the inlet technology, returns 540 instances). 

From the highest-level view, major corporate acquisitions and mergers seem to have slowed in recent years, and the rapid emergence of revolutionary technologies has morphed, with a few exceptions, into slower, evolutionary change. Agilent Technologies, with quadrupole and TOF technology, has joined the top tier and continues to promote chip-introduction schemes. Waters Corporation has followed suit with the advent of its TRIZAIC UPLC system. Waters' addition of ion mobility to its MS products provides the means to directly control the sample introduced into the mass spectrometer in an orthogonal fashion, layering a variety of ion manipulation techniques with differentiation of molecules by conformational cross-section. Thermo Scientific has steadily improved the speed of acquisition and resolution of its matched devices, the ion trap and the Orbitrap. The company's recently introduced dual-chamber ion-trap system, the LTQ Velos, begins to address some of the impediments inherent in ion-trap technology like space-charge and quantification limitations. 

Applied Biosystems today no longer exists as a corporation, surviving as a brand in the market as part of Life Technologies, a biotechnology manufacturer, after merging with Invitrogen (San Diego, California, USA). The company maintains its joint venture with MDS Analytical Technologies Sciex, which continues to improve the top-end, tandem quadrupole series. 

Based upon the same slanted attendance profile, we can, with some reservation, look at some year-to-year trends: 								

A data base query for ion suppression returns 69 instances representing unique abstracts, which seems typical (76 in 2008; 64 in 2007; and 87 in 2006). A disproportionately large amount of our analytical effort is based upon condensed phase practice, which is amenable to ESI. But ESI is prone to ion suppression and constantly requires better ways of remediation. 

Metabolomics at 139 hits shows a significant increase. In 2008, 80 was an increase from 46 in 2007 (39 in 2006, 54 in 2005). 

Biomarkers remains fairly constant with 355 instances versus 400 in 2008 and 350 in 2007. 

Informatics as a general term returns only 10 hits, while bioinformatics returns 154, down from 239 in 2008.

Making predictions is a tempting but risky exercise when you are committing to print. Yet in the 1997 article, I did venture to state that the "halcyon days of interface development were over," which appears to be true. A recent study report by Strategic Directions International, Inc., a market research firm that tracks instrument business trends indicates that, as of 2008, four manufacturers (Agilent, Applied Biosystems, Thermo Fisher Scientific and Waters Corporation) comprised 70% of the mass spectrometer market, with between 13% and 25% shares each. 

"Despite being well over a $2 billion market, demand for MS is expected to grow significantly, if not stronger than in recent years. This is despite current weakness in major economies the world over, as well as some pull back in the pharmaceutical industry. The fact that MS is such an important tool in high-end basic research in so many leading-edge industries will help to increase demand at a 9% annual rate through 2012. The market will be led for the foreseeable future by the more advanced methods, including Fourier transform (FT)-MS, tandem LC–MS, and quadrupole time-of-flight QTOF LC–MS."

The same report sees "typical" prospects for GC–MS settling for 5% growth, with half the business resulting from replacement sales of an installed base said to be reaching 30000 units. Improvements in GC-TOF and, to a lesser degree, GC–GC tandem account for increased sales. 

Often overlooked, even in these instances, is the central role single quadrupole instruments still play. A relatively large, installed base (7000 is estimated in this report), has Waters and Agilent competing closely, with equal shares dominating 90% of this market segment. 

QTOF instruments are reported to be at 4000 installed, proving, as I predicted in a 2004 column, that complexity coupled with improvements made this hybrid technology accessible for a variety of quantitative and qualitative applications.

Alan Millar (Senior Manager for QTOF Product Development, Waters) recently compiled an historical overview of the efforts of only a single company during the past decade. Eight nominal mass single-quadrupole instruments have been brought to market in the past 16 years, beginning with the Platform in 1993. A total of 11 triple- or tandem-quadrupole instruments appeared between 1989 and today, beginning with the Quattro model. Hybrid-TOF instruments account for eight unique product developments in 13 years, beginning with the original QTOF with 5000 resolution (FWHM) in 1996 to today's recent G2 introduction capable of 50000 resolution. Adding matrix-assisted laser desorption ionization (MALDI)- and TOF-only designs would easily more than double that number in a similar time span. 

Prices continue to be influenced by the cost of vacuum systems and control capabilities. Except for GC–MS, do not expect to see a high-end MS system for less than $100000. A counter-argument can be made that by any standard, single-quadrupole LC–MS instruments, which do sell typically for much less than $100000, offer state-of-the-art quadrupole technology today, lacking only the extensive additional capabilities of the more complex mass spectrometer. Median, or average, prices often appear in descriptions, except in the case of high-volume instruments (single-quadrupole and ion-trap instruments) and even those are often bundled in special arrangements. But without stating detailed specifications, you will find that the prices of higher-performing instruments overlap. 

As we have seen, technology can have a very brief half-life. Some make a great appearance only to fail to thrive after three or five years. Some, however, need enabling technologies to bolster their utility — TOF waited 60 years before fast electronics, software and much improved engineering design made them the practical analytical tool they are today. Software became more capable and hardware clearly improved in recent years, and an improved understanding of fundamentals often allows us to build on such improvements. 

A couple of recent introductions illustrate how sound fundamentals can reappear to our advantage. Both Bruker (Billerica, Massachusetts, USA) and Waters have recently commercialized an atmospheric pressure GC (APGC) adaptation for their LC–ESI-MS systems. The Waters license derives from DuPont and the work of Charles McEwen (currently Houghton professor of Chemistry and Biochemistry, University of the Sciences, Philadelphia, Pennsylvania, USA). McEwen is perhaps better known these days for his work developing yet another classic fundamental, the atmospheric solids analysis probe (ASAP). The ASAP technology allows an operator to simply place a solid or liquid sample on the end of a tube, insert the tube into a stream of heated nitrogen, where the sample is volatilized, and look at the resulting ions from the mixture. Direct links to Horning's work in the 1970s are readily apparent.

Debate over the exact nature and mechanics of ionization might still persist as it has with ESI while we made good use of it. But in the meantime, extremely fast chromatography demanded faster MS acquisition speeds with good fidelity. Improved software allows us to take advantage of our understanding of isotope characterization and our predictive capability. Better engineered, robust MS platforms displaying our improved understanding of gas dynamics yield sources capable of greater diversity. 

For instance, an aspect of the APGC Xevo design (Waters Corporation) puts a "mini-chamber" in place between the ESI inlet in the MS source and the GC transfer line as an integral part of the changeover from LC to GC. The entire procedure takes only 5 min. When the idea of LC and GC in the same instrument was proposed not too many years ago as a viable return-on-investment argument, it failed because substandard GC performance at any cost is unacceptable. Today we see low-femtogram sensitivity in what is ostensibly an LC source design. Add recent developments in atmospheric pressure photoionization (APPI), which show the potential to deliver EI-like spectra, and you realize that the same level of training and the same software and experimental design we've been accustomed to applying for LC–ESI-MS now covers a much more diverse chemical landscape. 

 is principal scientist, MS technology development at Waters Corp. (Milford, Massachusetts, USA); a former adjunct professor and visiting scientist at Roger Williams University (Bristol, Rhode Island, USA); co-founder and current president of the Society for Small Molecule Science (CoSMoS); and a member of 

Direct correspondence about this column to 

, Advanstar House, Park West, Sealand Road, Chester CH1 4RN, UK or e-mail: 

MS - The Practical Art

"In retrospect, we sometimes see the successful return of an earlier idea on the back of great advances in technology."

Commercialization of LC–MS: The Second Decade

Many of us use liquid chromatography (LC) methods supplied to us by others. These can be compendial methods — those that come from one of the pharmacopoeia — or they can come from the scientific literature or from another laboratory within our company. Such methods can be very specific in terms of the column and mobile phase to be used or they might be more general, so that the end user has some flexibility in adapting the method to his or her needs. One of the most common traits of such methods, however, is that the person who developed the method is not available to field our questions about the method. And the method does not usually have any background on why certain conditions were chosen. This month's "LC Troubleshooting" discusses the type of problems one can encounter when using such methods. 

The method in question specified a 150 mm × 4.6 mm, 5 µm particle C18 column (brand not listed) and a mobile phase of 57% methanol and 43% 10 mM ammonium carbonate buffer, pH 9.6, run at 1.5 mL/min. The column temperature was not specified. The sample is prepared by dissolving a drug tablet in water, filtering it and injecting 20 µL. The separation is simple, and with typical retention times of 2.5 and 3.7 min for the two components of interest, the requirement of resolution, 

, of more than 2 is met easily. However, the required tailing factor, 

, of  ≤1.5 is more of a challenge. Typically, for the second peak, 

 = 1.4–1.5 with a new column, but degrades to 

 > 1.5 after 1000–1500 injections. Installation of a new column solves the problem. I was asked how to fix this method. 

One of the first questions that I ask when presented with a problem method is whether the problem really relates to the method itself or if it is a problem with the LC system hardware. We can have the best method in the world, but if there is a problem with the LC system, we might not be able to use it. Fortunately, there is a simple way to check the system for reasonable performance. Just replace the column with a new C18 column and repeat part of the column manufacturer's column test. This can usually be accomplished by equilibrating the column in the mobile phase that was used to test the column, often 60–70% methanol–water run at a flow-rate of 1 mL/min. If you have the test sample, use it. Otherwise, nearly any well-retained neutral aromatic compound will work — toluene, methyl benzoate, naphthalene and so forth. The mobile phase should generate a peak that is retained with a retention factor, 

, of at least 2. The retention factor is calculated, as  

 is the retention time of the compound and 

 is the column dead-time (solvent front), normally determined by injecting an unretained compound, such as uracil or thiourea. You should observe a column plate number (also known as column efficiency) that is within 5–10% of the manufacturer's value and a peak that is nearly symmetrical (0.9 < 

Another way to be sure the system is working properly is to check its performance with a well-behaved method for another sample that you use regularly. Once you have checked out the system and it is determined to be OK, then you can point your finger at the method as the problem. Otherwise, you should correct any system problems before proceeding. In the present example, I recommended running a system check. For the remainder of this discussion, I will assume that it passed. 

After I am convinced that the LC system is working properly, I look through the method variables and list each item that might be causing the problem. Let us look at the possibilities: 

 The 150 mm × 4.6 mm, 5 µm particle diameter column is the workhorse of the LC industry and is about as trouble-free as a column can be. These columns are quite tolerant of pressure, sample size, flow-rate and other variables. When narrower diameter or shorter columns are used, extracolumn peak broadening can be an issue, so more care must be taken of the system plumbing and injection process. Smaller particles, while generating higher plate numbers, create more pressure and require smaller frits, which are blocked more easily. The 150 mm × 4.6 mm, 5 µm column is used widely at 1–2 mL/min flow-rates without generating excess pressure. So I doubt that the problem is related directly to the column size or flow-rate. 

 The C18 column is the most widely used column packing material for LC separations. However, the specific column brand and model were not identified. This is often the case for compendial methods, but less so for methods from the literature or for methods developed in-house. At one time we thought that all C18 columns were equivalent, but that assumption was discarded many years ago. For the present method, two major considerations come to mind. First, nearly all "modern" LC columns — those developed in the last 15 years or so — are based upon high-purity silica, often called Type B silica. These are more stable, less variable from column to column and less likely to have tailing problems than the older, less pure Type A silica columns. The distinction between Type A and B is not black and white, but is a continuum, generally (but not absolutely) with the newer Type B columns being of higher purity than the older Type B ones.  

Another characteristic of the higher purity columns is their stability at high pH. As a general rule, silica-based C18 columns can be used in the 2 < pH < 8 range without problems. Drop the pH below 2 and the bonded phase tends to hydrolyse. Above pH 8, the silica tends to dissolve. Some higher purity columns can be used at higher pH, say up to pH 10, but will be expected to have shorter column lifetimes than at lower pH values. Special "hybrid" silica columns are designed for use at pH > 8. These columns actually use less-pure silica — a well-controlled addition of methyl or ethyl functional groups within the silica structure provides for a more inert particle. Once bonded with C18 or other stationary phases, hybrid particles can be used to pH 10 or sometimes higher with normal column lifetimes. Another option for high pH work is to use a column packing comprising polymeric particles instead of silica. These are very pH-stable, but often do not work quite as well from a chromatography standpoint as silica-based columns, so they are less popular. 

Two red flags come to my attention with the stationary phase. First, is the silica one of the high purity or hybrid packings that can be used at pH > 8? Second, is the C18 bonded phase that was chosen sufficiently similar to the one used when the method was developed, so that the same separation can be obtained, especially in terms of the tailing factor? 

 As mentioned previously, most C18 columns work best when the mobile phase is 2 < pH < 8. The specified pH was 9.6, which is well above the normal region. This will usually result in shorter column lifetimes, with shifting retention times and degraded peak shape over time. Another potential problem with high-pH work is that some of the silanol (-Si-OH) groups on the silica surface will ionize. These ionic sites add ion exchange to the dominant reversed-phase retention mechanism, often resulting in peak tailing. This means that peak tailing at high pH often is more common than at low pH. 

To be effective, a buffer should be used within ±1 pH unit of its p

 of carbonate is 10.3, so buffer pH should not be an issue per se. However, ammonium carbonate is a volatile buffer. This makes it a common choice with mass spectrometry (MS) detection, or other evaporative detectors, because the mobile phase must be removed by evaporation. However, the volatility of carbonate buffer reduces its long-term stability, so many workers make fresh buffer every day to minimize buffer-related changes to the mobile phase. This might come as a surprise to users accustomed to phosphate buffers that are often used for 1–2 weeks without concern. Methanol is one of the two most common organic solvents used in reversed-phase LC today, so it is unlikely the source of the problem.  

I am concerned about the age of the buffer and the effect of the high pH on column stability. 

 Those of you who read this column regularly know that column temperature is one of my "hot buttons". For isocratic separations, retention times can vary by 1–2% for each 1 °C change in temperature; the change is less dramatic for gradient separations, but is still important. In addition to retention shifts, selectivity often changes with temperature changes, especially if ionizable analytes are present. A change in temperature can change the mobile phase pH, the ionization of the analyte and the retention time; the combination of these can wreak havoc on reproducibility. Even if the laboratory temperature appears to be well-controlled, according to the thermostat, the microclimate containing the LC system can be much more variable, especially if a heating or air conditioning duct blows at the system. Operating the column at ambient temperature can generate some very surprising results when a method is transferred. Last year in January I visited a laboratory in China that was 10 °C and one in Tel Aviv in June that was 30 °C. Both used ambient column temperatures, but if our 2%/°C rule is applied, the retention times could vary by 20 °C × 2%/°C = 40%! I'm sure that the temperature of the laboratory in question does not change this much. 

In spite of my concern about control of the column temperature, I don't think it is related to the current problem. The resolution is acceptable and peak tailing is unlikely to be affected strongly by normal changes in ambient temperature. 

 The sample preparation in the present method is very simple and there isn't much to go wrong. It is usually preferred to inject the sample with the mobile phase as a sample solvent, but with an injection volume of only 20 µL, this is unlikely to be a problem. So I wouldn't be concerned about this aspect of the method. 

 The retention time of the first peak is 2.5 min. We need to translate this into a 

-value for a qualitative assessment. For this we need a value of 

 from the column test performed earlier. Or when 4.6 mm i.d. columns are used, we can estimate the column volume as another option. The volume (in milliliters) is approximately 0.01 

 is the column length (in millimetres). The 150 mm column's volume is ≈ 1.5 mL. This is converted to 

 by dividing by the flow-rate (1 mL/min) for a value of 

 = (2.5 – 1.5)/1.5 = 0.67. For the second peak 

 = (3.7 – 1.5)/1.5 = 1.5. As a general guideline, I like to see 

-values of at least 1 and preferably 2 or more for the first peak. Shorter retention times tend to cause the peak to be coeluted with interferences that come out at the solvent front and early peaks are much more susceptible to extracolumn effects and injection problems that can cause peak tailing. So it is possible that the short retention time might be contributing to the tailing problem. It would be easy to test this by reducing the organic content of the mobile phase by 5% from 57% methanol to 52%. This should increase retention, and if this were the problem source, would be an easy fix. Note that the short retention might not be due to a flaw in the method, but by using a different column than that used to develop the method. Different brands of C18 columns can cause retention times to differ by a factor of two or more under nominally identical conditions, although smaller retention changes are more common. 

I don't like the small retention time of the first peak. This might or might not be the problem source, but would be easy to check by a simple mobile-phase adjustment. 

 The user implied that the method was acceptable for the first ≈ 1000 injections before the tailing became too large to pass the system suitability test. Although column lifetimes of 2000 injections or more are common with simple drug product samples, such as this, 1000 injections is not unreasonable. If you consider the cost of a column, for example, $500, distributed over 1000 injections, it is only $0.50/injection for the column. In most laboratories, when all costs are considered, this is likely to be less than 1% of the cost of the analysis. At this point, the column has paid for itself and could be replaced without a significant impact on the cost of analysis. The concept of the column as a consumable item might seem strange at first, because of its high purchase price, but when compared to other consumable items in the chromatographic process, such as solid-phase extraction cartridges and even solvents, the column is not expensive on a per-sample basis.  

I would like to see a longer column lifetime than was observed, but especially under the high-pH conditions, where column life is expected to be shorter, it might not be a major concern. 

We've made our list of potential problems in the system, now we need to figure out how to correct the problem. The potential problems cluster around the combination of the column and mobile phase. The high pH is expected to shorten the column life when compared to pH < 8; even the hybrid columns are expected to have shorter column life at pH > 8. As the column ages, the surface chemistry changes and peak shape changes often result, so increased tailing is not surprising. I would make sure that I was using the same column brand and model as was used in the original method, if this information is available. Otherwise, a high-purity silica or hybrid column would be the column of choice. Certainly the column temperature should be controlled, even though it is unlikely to be the problem in the present case. 

Premature loss of buffer capacity due to the volatile buffer is easy to check by making a fresh batch of buffer — this is probably the first variable to check, because it is a simple test and if successful, could be incorporated in the routine use of the method. If a fresh buffer does not help and if a change in column does not correct the problem, or if the correct column is already in use, it might be worthwhile making a change in the mobile phase strength to see if larger retention times helps reduce the problem. Of course, this change might not be permissible for routine use of a validated method, but it could be tried on a one-time basis to see if it helped.  

If this were my method, I would probably make a cursory evaluation of the variables, such as checking on the column specification and making up a new batch of buffer. If these did not solve the problem, I would live with the problem and replace the column more often. In the long run, it will be much less expensive to replace the column regularly than to make an involved investigation that might end in requiring revalidation of the method. The time and expense involved is probably not a good investment in most laboratory settings. 

 is vice president of LC Resources, Walnut Creek, California, USA; and a member of the Editorial Advisory Board of 

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