In an astonishingly short time, mass spectrometer manufacturers have developed highly capable, compact instruments that integrate well with advanced liquid chromatography (LC) systems. Driving that achievement are breakthroughs in technology and manufacturing design, not to mention a universal desire to seek out and serve practitioners' needs. Consequently, the choice of instruments ranges widely. Some exotic hybrids offer ultrahigh resolution and confer novel benefits like ion mobility and a variety of interfaces. Others are low-resolution instruments that, despite their relatively low cost, nevertheless display capabilities akin to the best-performing instruments of a decade ago. Yet there might be another option on the horizon if the application is understood well enough and can justify the narrow definition of a non-LC or, perhaps, limited-LC introduction system.  

In previous columns, we made glancing references to what we might lump together as nonanalytical uses of mass spectometry (MS) in airport security applications and for process instruments. In some cases, because of its greatly reduced vacuum requirements, a gas chromatography (GC)-only design edges analytical chemistry closer to a design that is less costly but still capable in the MS model. Relatively recently, two companies have emerged to raise interest in small, portable mass spectrometers. Simultaneously, some complementary technologies, such as thermal desorption, have excited interest because full sample preparation and LC separation are not required routinely. 

So Why Isn't Every Bench Home to a Mini-MS System?

A frequent impediment to developing a new instrument of any kind is cost and expected market return. Startups may find it easier, at least initially, to introduce novel technologies than larger corporations because they often are burdened with less diverse financial demands and often focus resource in a singular product definition. The longer view of market potential eventually determines whether the technology becomes a commercially viable product, as opposed to a curiosity destined to become an historical footnote. For instance, the less selective instruments requiring high acquisition threshold settings employed today in airport security could be replaced by more selective mass spectrometers. Yet, for a market viewed as finite (estimates are of fewer than about 10,000 units, total), the level of support required, including training and upkeep, mitigates against development. 

Nevertheless, two smaller companies, Torion Technologies, Inc. (American Fork, Utah) and Griffin Analytical (a business unit of ICx Technologies, West Lafayette, Indiana), are pursuing a broader vision. Both support small ion-trap technology using different basic approaches that fit applications such as illegal drug detection and other homeland security uses. A small suitcase-sized unit from Torion, the GUARDIAN 7, has illustrated its GC capabilities analyzing security samples and toxic industrial chemicals (1). The GUARDIAN 7 can sample directly from a solid-phase microextraction (SPME) membrane. Similarly, the Griffin design can acquire data from a sorbent-captured sample which is then brought to the MS. The 600 model from Griffin — while not exactly miniature — runs completely unattended. 

Meanwhile, some researchers have forged ahead, relying on the recent and easily accessible return of thermal desorption called "ASAP" (atmospheric solids analysis probe). ASAP is a product of M&M Consulting (Hockessin, Delaware), which has commercialized it to fit a wide range of mass spectrometers. A probe design manufactured by Waters Corporation (Milford, Massachusetts) incorporates the ASAP technology suited for the entire current line of MS instruments. When this probe first appeared on the market, its potential and fundamentals were discussed in peer-reviewed journals and in this column (2–4). Apart from its commercially engineered advantages, the technology harkens back to early work by Horning and others. Horning exposed a liquid or solid sample placed upon an inert, solid probe to hot gas in the presence of ions. Thus, he created ions of interest (5). An oft-used diagram (Figure 1) indicates why a simple process that involves atmospheric pressure chemical ionization (APCI) (and perhaps atmospheric photoionization [APPI]) and the readily available nitrogen stream within an instrument's source works surprisingly well. Many samples from a variety of liquid and solid matrixes once volatilized are amenable to ionization by APCI and APPI without first diluting or otherwise preparing the sample.  

APCI overlaps much of what we consider the domain of electrospray ionization (ESI). ESI has been central to our practice for many years. Our thinking tends to be so "LC-centric," because we analyze polar compounds with it (as well as those that ionize readily from a reversed-phase solution) after separating or at least dissolving samples in solvent. So what many discover when they first use the ASAP probe is its sensitivity. Because we are conditioned to LC introduction, which requires us to dilute samples, users might be shocked at its apparent sensitivity. The sample is used directly, often in its final finished form, undiluted, and so at much higher concentration. 

Two articles published in 2004 and 2006 summed up the advantages and disadvantages of ion-trap technology (6,7). Because portable mass spectrometers require reduction in size, weight, and power consumption, important components like vacuum pumps must also undergo miniaturization. The ion-trap mass analyzer design is a good place to start. Inherently small, it operates with few ion-optic elements, which do not require a highly precise alignment (unlike other types of mass analyzers) to achieve their specifications. You can perform multiple stages of MS (fragmentation) in the same mass-analyzer space of these trapping devices. Moreover, the operating pressure for ion traps can be higher than that required for other forms of MS, making pumping requirements less stringent (6): 

"(R)adio frequency . . . trapping voltage is inversely proportional to the square of the analyzer radial dimension, a modest decrease in analyzer size results in a large reduction in operating voltage and, hence, lower power requirements. An added potential benefit of the reduced analyzer size is the shorter ion path length, which may ease the vacuum requirements even further. This is an especially important area as some of the most limiting aspects of MS miniaturization are not in the ion optic components, but rather in the vacuum and other support assemblies." 

The Lammert paper (6) addresses a downside of ion traps as the basis for the device: 

"The ability to miniaturize ion trap mass spectrometers hinges on addressing the issues of space charge (8) and machining tolerance limits. Miniature ion traps with conventional ion trap geometries (i.e., hyperbolic surfaces) have been previously explored (9). However, as devices become smaller, the machining tolerances play an increasingly significant role in trapping field defects." 

The limitations of space charge, significant for ion traps, are handled in various ways in larger commercial instruments. When a certain well-characterized number of ions is exceeded in a given space, the measured results (mass calibration and linearity response) are no longer dependable. 

The toroidal design of the Torion instruments offers some advantages in miniaturization illustrated here over attempts so far (Figure 2), especially with 2D designs developed to overcome space charge effects or increase ion capacity on commercial instruments.  

Because both Griffin and Torion came into existence in 2001, sufficient time has elapsed to monitor how their technologies have performed, which until now have been predominantly GC-based analyses. Looking forward, Garth Patterson, chief technology officer at Griffin, takes a longer view. While technology and manufacturing issues are being resolved, the future lies in developing sample introduction (10) such as desorption electrospray ionization (DESI), direct analysis in real time (DART), and others.  

Graham Cooks' group at Purdue (Purdue University, West Lafayette, Indiana), DESI was developed, gives us a view of how thermal desorption techniques might solve problems attendant with coupling them with miniaturized designs. A recent paper from the Cooks group sheds some light on how the limited stage design of a mini-MS might achieve improved sensitivity and linearity through discontinuous atmospheric ion introduction (DAPI) (11). The small, portable design referred to as the Mini-11 weighs a mere 5.0 kg (11 lb) with batteries. The device, which consumes less than 35 W, is only 12 cm wide and 22 cm long.  

I recently spoke with Gary Van Berkel, group leader and distinguished researcher of the Organic and Biological Mass Spectrometry Group at Oak Ridge National Laboratory (Oak Ridge, Tennessee). From the ORNL perspective, he characterized attempts made over many years to develop field-portable MS instruments for security as a holy grail-like quest. He said that people want research-MS capabilities at reasonable cost and, significantly, without consumables, which can dampen interest. Beyond what we might consider consumables in the laboratory, a required gas or liquid supply and dependence upon lasers or other high-level components renders the idea less attractive in the field. 

Because of its low cost, ASAP fits the "no consumables" requirement more closely than some other thermal desorption techniques (Figure 3). Though it does require some form of heating (hot nitrogen suffices), the sole consumable is a melting-point capillary (perhaps even a reusable one, at that).  

In a presentation at IMSC (Bremen, Germany) on August 30, 2009, Richard Fussell reported work underway at the Food and Environmental Research Agency (FERA, York, UK). FERA is investigating ways to test agriculture fields for contamination with chemicals like fungicides, which are used frequently in agriculture but which should not find their way into our food supply. Direct interrogation of the site as it stands with minimal sample handling is an attractive goal. Sample portability and protection of the sample's integrity "as sampled" before analysis must be assured and expectations of sensitivity and response must be met. The FERA group reported that 'field incurred' residues of strobilurin pesticides could be detected by simply stirring the ASAP probe directly into grains of wheat. Further research in the use of ASAP (or ambient ionization techniques if you prefer) is ongoing as part of the European Project on the use of new technologies for screening multiple contaminants in food. For more detail see the BioCop project (

Jackie Mosely, senior research officer in the chemistry department at Durham University (Dunham, UK) is also considering alternative means of employing the selectivity and specificity of MS. (Incidentally, Mosely's website is a good source of related information. Go to 

, and click "Dr. Jackie A. Mosely.") In a recent conversation, she drew from her experience with Bruker Corporation (Billerica, Massachusetts) as well as her research and commented on three areas of interest. First, in research: 

"The prospect of bringing a small MS into a specific, conditioned environment, such as a glove box, to handle compounds like pyrophoric chemicals, which are otherwise combustible in atmosphere, is attractive. Today our only choice is to build a glove box around the MS."  

As her second case, Mosely points out the need for a simple sample introduction method: 

"Synthetic chemists need only low resolution MS to quickly confirm their efforts. Without a utilitarian (mass spectrometer), equipped with something like an atmospheric insertion probe, they are required to do full-fledged sample prep, plus LC introduction for MS."  

Finally, for her third case: "A small (mass spectrometer) would be a monumental advantage to teaching. The ability to introduce a sample easily to a small, portable (device) allows students a direct connection to being taught the fundamentals of chemistry."  

A Quick Assessment Tool for the Synthetic Chemist

Peter J. Lee is a principal application chemist at Waters Corporation who holds an M.Eng. degree in organic synthesis from Kyushu University, Japan, and a Ph.D. in chemistry from Emory University (Atlanta, Georgia). With 50 articles and six U.S. patents, his research interests have included developing analytical methods used in food safety, environmental, pharmaceutical, and chemical analysis. 

As an example of ASAP utility for synthetic chemists, Lee demonstrated a commonly used synthesis where a direct analysis might apply: 

"In the chemical industry, the principal job of an organic chemist is to develop new chemicals and increase the production yield. In many cases, developing a new product or a new process with only a 5% increase in the yield can result in earnings of millions of dollars for (a) company. Organic chemists spend the majority of their time setting up reactions, monitoring the progress of reactions, working up reaction mixtures, and purifying products. They need to quickly obtain chemical structure data for making the right decisions about when to stop reactions and which fractions to pick to obtain products when monitoring reaction workup and purification processes." 

Time-consuming and labor-intensive sample workup procedures such as extraction, filtration, separation, and evaporation are typically required before analysis. Thin-layer chromatography (TLC) often is employed, which does not yield the specificity of MS (at least not without added steps). Chemists, whose expertise lies in analyzing new reactions and new compounds, in developing methods, and in data interpretation, must often wait hours for the data when using conventional tools such as nuclear magnetic resonance, LC–MS, or LC. Highly desirable technologies are those that can provide critical structure information quickly so that chemists can make informed decisions. 

To illustrate the utility of ASAP as a direct means of interrogating a reaction without first preparing a sample, a reductive amination reaction based upon a well-referenced paper (12) was conducted. The reaction's progress was monitored using ASAP together with a small, tandem-quad mass spectrometer. Reductive amination is widely used in multiple-step organic syntheses for making agricultural chemicals, food additives, and building blocks for high performance materials (Figure 5). According to the paper, this reaction was typically run for 48 h, followed by approximately an hour-long reaction workup procedure. When analyzing this reaction mixture by LC or LC–MS methods preparative procedures are required to remove sodium cyanoborohydride (NaBH

CN), the reducing agent, which otherwise would plug ESI capillary tubes and ruin LC columns. Using ASAP, the reaction mixture was loaded directly onto the sealed-glass, melting-point capillary tube of the probe for MS analysis, without workup or sample cleanup.  

The ASAP probe was inserted into the sealed source enclosure, and the desolvation gas was heated rapidly to 400 °C. Data acquisition was 30 s, in positive mass scan mode. The source temperature was 120 °C, and the desolvation gas flow 500 L/h. Combined mass spectra were obtained by subtracting the baseline of the reference scans from the samples' total ion current profiles.  

The reaction (Figure 6) was monitored by following the disappearance of the starting materials: 									

The spectrum in Figure 6a was acquired after mixing acetophenone (AP) and aniline (AN) in methanol.

The spectrum in Figure 6b was acquired 2 min after adding acid and shows the formation of an imminium intermediate (IM).

The spectrum in Figure 6c, acquired 10 min after adding the reducing agent, shows the formation of the product, phenyl(phenylethyl)amine (P) and the disappearance of the imminium intermediate.

The spectrum in Figure 6d, acquired 40 min after adding the reducing agent, shows the increase of the amine product (P) and the decrease of the starting reactants (AP and AN).

The spectrum in Figure 6e was acquired 4 h after adding the reducing agent and reveals no remaining reactants, indicating the completion of the reaction.

Just a few years ago, the ability to do away with sample preparation was touted — perhaps overoptimistically — as hope for the future of atmospheric thermal desorption. As often happens when a technology comes of age and is applied to solving real problems, the end result is far better in combined use with other technologies. A small refinement to this process shows that sample prep is not always a chore best avoided. For example, an acetic anhydride reaction benefits from a quick preparative cleanup, thus dispensing with the burden of trying to characterize a complex MS spectrum that had not first undergone LC separation.  

Amide formation is one of the most important reactions in organic synthesis. In this case (Figure 7), Lee set up a reaction by mixing methyloxybenzylamine (A) with base catalyst 4-dimethylaminopyridine (C) in CH

CN and adding acetic anhydride to produce acetamide. The spectrum in Figure 8b indicates the reaction was complete in 5 min. The spectra in Figures 8c and 8d show the product, acetamide, after a simple nonquantitative purification (PoraPak Rxn CX cartridge, Waters Corporation) to enhance the presence of ions of interest.  

Articles

MS- The Practical Art

With the advances in LC-MS in recent years, is there another practical option on the horizon for those who need the specificity of MS and can justify the narrow definition of a purpose-built system?

Could This Be Your MS System of the Future?

Peptides perform a wide variety of physiological functions and are critical to regulatory and cell signaling pathways. As such, many peptides have been investigated as therapeutic drugs and are active vasodilators, vasoconstrictors, hormones, and neuropeptides. Any peptide under consideration as a therapeutic drug must be analyzed to determine both the peptide content and any counterions that might be present. 

Oxytocin is a neuropeptide with therapeutic uses including stimulating labor, control of postpartum hemorrhage, and inducing lactation (1). This nonapeptide has a disulfide bond between two cysteine residues forming a large ringed structure (Figure 1) that is stable between pH 3 and 5. In more acidic solutions, the peptide hydrolyzes, whereas in neutral and basic conditions it produces dimers and polymeric mixtures by the creation of intermolecular disulfide bridges that lead to peptide deactivation (2).  

As a commercial product, oxytocin is available as an acetate salt. The USP and EP allow 6–10% by weight of acetic acid in the solid peptide drug. The presence of acetic acid as a counterion in peptide therapeutics is common and is found in numerous small peptides. Current USP and EP monographs use two separate methods to assay oxytocin and the acetate counterion (3–6). The USP monograph to assay oxytocin uses a gradient of sodium phosphate and acetonitrile on an L1 (C18) column with a 20 min run time and a flow rate of 1.5 mL/min. The EP oxytocin assay monograph is similar to the USP monograph, in terms of the column type and mobile phase. However, the EP method specifies a 1.0-mL/min flow rate and a 30-min run time with an additional 15 min of equilibration. The current USP and EP chapters for acetic acid in peptides are equivalent and are not specific to acetic acid in oxytocin. Like the oxytocin assay method, an L1 column is used, in this case with a phosphate buffer–methanol gradient. This acetic acid method has a run time of 22 min at a flow rate of 1.2 mL/min and uses a steep gradient to wash the peptide from the column after the acetate is eluted. To fully analyze oxytocin acetate by the USP methods takes a minimum combined total of 42 min and two separate injections with different methods. 

Acetic acid is retained weakly on an L1 column and quantification can be challenging, particularly if other compounds that elute near the void volume are present. Because of this weak retention of acetic acid, the rapid simultaneous determination of oxytocin and acetate in a single injection on an L1 column is difficult. Chromatographic conditions that retain both acetic acid and resolve the peptide and related impurities lead to long run times compared to current high performance liquid chromatography (HPLC) methods. Alternative methods to assay neuropeptides such as enzymatic assays, spectroscopic methods, gas chromatography, capillary electrophoresis, mass spectrometry, and ion chromatography effectively measure either the peptide or the acetic acid, but have inherent difficulties in simultaneously determining the peptide and an acetate counterion (7–9). 

Conversely, HPLC separation on a mixed-mode weak-anion-exchange column retains acetate, allowing better quantification of the low UV absorbing acetate ion and also retains the peptide of interest. By using pH to control the selectivity of the column, a single method can be developed for simultaneously determining peptides and acetate counterions (10). 

 Oxytocin, angiotensin I, bradykinin, neurotensin, and tetra-sodium pyrophosphate decahydrate were purchased from Sigma-Aldrich (St. Louis, Missouri). Potassium phosphate, monobasic was purchased from Fisher Scientific (Hampton, New Hampshire). Acetonitrile, chlorobutanol, methanol, phosphoric acid, and pH buffers were purchased from VWR Scientific Products (West Chester, Pennsylvania). Glacial acetic acid was purchased from USP (Rockville, Maryland). Deionized water, type 1 reagent grade, 18 MΩ-cm resistivity or better was used throughout. 

 Aliquots (25 μL each) of sample were injected onto an UltiMate 3000 HPLC system (Dionex Corp., Sunnyvale, California) with a mixed-mode weak-anion-exchange reversed-phase column (150 mm × 2.1 mm, 5-μm Acclaim Mixed Mode WAX-1 (Dionex Corp.) at a flow rate of 210 μL/min. The peptides were eluted with a 36-min gradient as follows: 98% 50 mM potassium phosphate at pH 4.2 (A), 2% methanol (B) for 5 min, 2–50% B in 20 min, 50% B for 10 min, 5 min of equilibration consisting of 2% B before injection. The column temperature was maintained at 30 °C. Detection of eluted peaks was accomplished by a variable-wavelength UV–vis detector set to 220 nm. 

The USP monographs for oxytocin and acetic acid in peptides were repeated using a 150 mm × 2.1 mm, 5-μm Acclaim 120 C18 column (Dionex Corp.). 

 Acetic acid standards: A 20.0-mg/mL stock solution of acetic acid was prepared and stored at 4 °C. Working standards were prepared daily from the stock solution. 

Peptide samples: Peptide stocks were prepared by dissolving 1.0 mg of peptide in 1.00 g of deionized water. These stocks were further diluted to 250 μg/mL with mobile phase A before analysis. Stocks and samples were stored at 4 °C. 

Oxytocin with chlorobutanol: 250 μL of a 1.0-mg/mL stock solution of oxytocin was diluted to a total volume of 1 mL in 95:5 50 mM potassium phosphate (pH 6.0)–methanol containing 0.5% chlorobutanol. 

 Figure 2 shows an example of the separation of acetic acid and oxytocin using the USP oxytocin assay (3). Acetic acid is retained weakly on the column under the conditions used for the oxytocin assay. Quantification of acetic acid at concentrations present in the peptides would be very difficult, particularly if there are other poorly retained components in the drug formulation. The chapter method to determine acetic acid in peptides has better retention of acetate, but can be prone to interference, as shown in Figure 3. The acetic acid present in the peptide is difficult to determine due to a closely eluting peak from an impurity in oxytocin. Spiking the sample with acetic acid confirms the presence of acetic acid and further illustrates the difficulty in quantifying acetate in peptide samples. The acetic acid method cannot be used to quantify oxytocin due to low resolution between the peptide and neighboring peaks.  

In comparison, Figure 4 shows the separation of acetic acid and oxytocin in a peptide sample on a mixed-mode column. The pH of the mobile phase was optimized at 4.2 after investigating a range of 3–6. A pH of 4.2 offers the best compromise between resolution of peptide impurities, acetate retention time, and total run time. At pH values below 3.5, acetate is retained weakly on the column and pH values of 5–6 caused poor resolution between oxytocin and peptide impurities. At the optimized pH of 4.2, the acetate and oxytocin peaks are resolved from other impurities and easily quantified. The resolution between oxytocin and the next nearest peak is 2.6, greater than the USP monograph requirement of 1.5. A peptide impurity, most likely small amounts of the oxytocin dimer (peak 3), is present in this peptide sample that is eluted at 27 min. If this impurity is absent, the run time can be reduced to 20 min.   

Chlorobutanol is a common preservative that is approved for use in injectable oxytocin solutions. To confirm that the presence of this preservative does not interfere with oxytocin or acetate quantification, an oxytocin sample was prepared with chlorobutanol. There is significant resolution between oxytocin and chlorobutanol (USP 

 = 18.16). The USP resolution between oxytocin and the next nearest peak is equivalent to the resolution in the unpreserved sample. The chlorobutanol, with a retention time of 25.23 min does not interfere with determination of acetate or oxytocin.  

Quantification Assay Linearity, Limits of Quantitation, and Limits of Detection

 Determination of oxytocin by the EP method currently requires 250 μg/mL of oxytocin (4). The USP monograph assay specifies 10 USP units/mL, which is equivalent to 25 μg/mL of oxytocin assuming an activity of not less than 400 units/mg (3). The linear response of oxytocin determination by detection at 220 nm was investigated from 15 μg/mL to 250 μg/mL to cover the range of both monographs. The correlation coefficient across this range was determined to be 0.9997.  

The LOD and LOQ for oxytocin determination were measured by injecting samples that produced a response three and 10 times the measured peak-to-peak noise, respectively. These injections confirmed the estimated LOD of 0.06 μg/mL and estimated LOQ of 0.2 μg/mL for oxytocin. These values are well below the concentrations specified by the EP and USP, ensuring straightforward quantification.  

Table I: LOD, LOQ, and linearity for the determination of acetate and oxytocin 

 The concentration of acetate counterion in peptide preparations can vary greatly between peptides. The linearity of the acetate response was tested between 5.0 and 35 μg/mL to cover the expected range of acetate in the peptides investigated. The correlation coefficient, LOD, and LOQ are presented in Table I.  

Table II: Determination of acetate in four different peptides 

Precision and Accuracy of acetate analysis in representative peptides:

 The amount of acetate present in the oxytocin sample used for this work is small compared to the amount allowed in the drug product. Containing ~0.5 moles of acetate per mole of oxytocin, the weight percent of acetate is only 2.5%. However, the amount of acetate in peptides can be as high as 10% (w/w). To determine the concentration of acetate in peptide solutions, four peptides were prepared at 250 μg/mL: angiotensin 1, bradykinin, neurotensin, and oxytocin. These peptide samples were each analyzed to determine acetate. Table II presents the peptide precision and acetate determination data for the four representative peptides that incorporate acetate counterions. In all cases, the peptide peak area precisions (RSD) are <0.7, which exceeds the USP criteria for oxytocin. The acetate peak area precisions (RSD) are all <2, exceeding the USP and EP criteria of <5.  

Figure 5 illustrates the separation of acetate from the peptides for oxytocin, bradykinin, angiotensin I, and neurotensin. In each case, the acetate and the peptide are quantifiable with good resolution of peptides and impurities using a single injection. Figure 6 shows an expanded view around the acetate peak. The acetate peak is integrated easily with no interferences from other components in the peptide samples.  

Recovery of acetate in the four peptides was tested by spiking acetic acid into the peptide solutions, except neurotensin, which was spiked with sodium acetate. The recoveries ranged from 98% to 105% (Table III). These data suggest that the determination of acetate in peptide samples is accurate. Comparison of the lot analysis of acetate in the peptides to that determined is also shown. The determined acetate amounts in the peptide samples are in agreement with the labeled acetate to peptide mole ratio. The concentrations are within ±10% of the label claims or the lot analysis. The neurotensin sample contains 8% acetate, as measured by ion chromatography, based upon the certificate of analysis for the lot. The determined amount of acetate in neurotensin was consistent with the value of 8% acetate, as listed in Table III. Both recovery data and determined acetate amounts in comparison to the suppliers stated amounts show the method to be accurate.  

Table III: Recovery of acetate in peptide preparations 

The mixed-mode column has been used to determine peptides and the acetate counterion in a single injection simultaneously. The method is fast, accurate, and has the sensitivity needed to determine acetate concentrations in 250 μg/mL peptide samples. The resolution and precision for replicate injections exceed the USP and EP monograph requirements for both oxytocin and acetate. Acetic acid has been quantified successfully in four different peptides, each with a different native acetate concentration, showing the flexibility and broad applicability of this method.  

Deanna C. Hurum, Brian M. De Borba, and Jeffrey S. Rohrer

Dionex Corporation, Sunnyvale, California. 

(1) C.M. Karbiwnyk, K.C. Faul, S.B. Turnipseed, W.C. Andersen, and K.E. Miller, 

(3) Oxytocin, The United States Pharmacopeia 31, American Pharmaceutical Association, Washington, DC (2008).

(4) Oxytocin, EP 5.0 01/2005:0780 The European Pharmacopeia, 5th ed., Council of Europe (2005). 

(5) Acetic Acid in Peptides <503>, USP 32-NF27, American Pharmaceutical Association, Washington, DC (2010).

(6) Acetic Acid in Synthetic Peptides, EP 2.5.34 The European Pharmacopeia, 5th ed., Council of Europe (2005). 

(9) I.N. Glazkov, N.L. Bochkareva, I.A. Revel'skii, 

(10) Dionex Corporation. Simultaneous Determination of Pharmaceutical Peptides and Acetate by HPLC with UV detection using the Acclaim Mixed Mode WAX-1 column, Application Note 234, LPN 2291. Sunnyvale, California (2009). 

Article

Oxytocin acetate samples were analysed successfully for both the peptide and acetate with a single method using a mixed-mode weak-anion-exchange column.

Simultaneous Determination of Synthetic Pharmaceutical Peptides and the Acetate Counterion by Mixed-Mode Weak-Anion-Exchange Chromatography

Pittcon 2010, referred to in its full name as the 61st Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, returned to the massive Orange County Convention Center in Orlando, Florida, from February 28 to March 5, 2010. This year's event hosted 960 instrument manufacturers and 1aboratory suppliers in more than 2000 booths. In addition to attending the exposition, the conferees listened to 2000 technical presentations, checked numerous company seminar rooms, or attended one of 140 short courses. 

Undoubtedly, Pittcon still remains the most important yearly international analytical exhibition, where companies introduce their latest instruments, instrument accessories, software, columns, sample preparation, and other consumable products. Because many past attendees have purchased one or more new products within three months after attending the show, most exhibitors attempt to maximize their booth traffic to meet as many potential customers as possible. 

The purpose of this report is to provide information about many of the new separation consumables and accessory products that will be displayed at Pittcon 2010. In some cases, products that were introduced during 2009 but after Pittcon 2009 (1,2) may be included for reasons of completeness. The information is based upon manufacturers' responses to a questionnaire mailed in early 2010. Because of space limitations and the fact that some manufacturers did not respond to the questionnaire, this report cannot be considered an exhaustive listing of all new products that were introduced in Orlando.  

However, over the years, these Pittcon introduction summaries have provided a good source of information that would be difficult for one individual to gather during the four days of the exhibition. In addition, the products introduced have shown definite correlations to current research, development, and application activity in the separation sciences. 

As in previous years, columns and other products recommended by their manufacturers primarily for biomolecule separations or sample preparation are denoted in the tables with the designation BIO. Some of these products might be used for general high performance liquid chromatography (HPLC) separations as well, but their main emphasis is for biological samples.  

In last month's coverage, I described new introductions in the areas of HPLC and reversed-phase, normal- and bonded-phase, ion-exchange and ion, size-exclusion, large- and preparative-scale, and specialty chromatography columns. This month, I will look at gas chromatography (GC) columns, sample preparation products, and hardware, accessories, and small tabletop instruments, mainly for sample preparation. 

Although GC is considered to be a relatively mature separation technique, new GC columns continue to be introduced each year at Pittcon (Table I). This year introductions were rather sparse compared to previous years: a new family of columns, six new individual columns, and a guard column were introduced. The GC columns were all wall-coated open tubular (WCOT) format. 

In line with recent introductions, Thermo Fisher Scientific (Madison, Wisconsin) introduced a new family of GOLD GC columns that are their low bleed, inert capillary products. The brand name GOLD also has been used for their improved line of ultrahigh-pressure liquid chromatography (UHPLC)/HPLC columns. 

Applications-specific GC columns are always popular introductions at Pittcon. This year, a majority of the columns were for specific application areas. In the environmental domain, columns for the U.S. Environmental Protection Agency's polynuclear aromatic hydrocarbons (PAH) were introduced by Varian (Palo Alto, California) and Agilent (Santa Clara, California). The Varian column has a proprietary PAH selector that was designed to optimally separate the difficult pairs, including the chrysene and triphenylene peaks. The Agilent column was designed to meet the EU PAH method so each column is tested to ensure separation of the compounds in their mixture. Other applications-specific GC capillary columns introduced at Pittcon included those for detailed hydrocarbon analysis and U.S. EPA priority pollutants. 

Gas chromatographers are always asking for their columns to do more so Phenomenex (Torrance, California) has launched additional phases in their high-temperature Inferno line of GC capillary columns. Normally, when columns are pushed to 400 °C, metal columns are used. Phenomenex stays with a polymeric coating for the fused-silica capillary housing that allows them to be used at that temperature. The higher temperature capability allows the columns to analyze even higher boilers or permits the column to be baked to remove high boiling contaminants that could bleed and cause problems. 

As indicated in Table II, Pittcon 2010 was another productive year with 21 new sample preparation products or families of products displayed. Not counted were sample preparation accessories (Table III), which will be covered later. Among the more popular introductions were products to deal with increasingly smaller amounts of available sample. Many solid-phase extraction (SPE) products ranging from small mass cartridges to pipette-tips to 96-well plates were among the formats introduced. Just about every mode of SPE (reversed phase, strong anion, and cation exchange, adsorption–normal phase, complexation, mixed mode and hydrophilic interaction liquid chromatography [HILIC]) was representative. A wide variety of media were introduced at Pittcon including polymer-based, carbon and graphitized carbon black, silica-based, and a number of other inorganic oxides (for example, ZrO

About half of the SPE products introduced at Pittcon 2010 were polymer-based. Polymeric SPE products are particularly attractive because they can dry out during the conditioning steps without affecting the recovery and reproducibility of the SPE experiment. They also can be treated with more aggressive washing solvents than silica-based sorbents because their pH range is greater. Another advantage of the polymeric SPE sorbents is that they often display mixed mechanisms and multiple interactions, which can be used to retain analytes or matrix compounds selectively. Because the surface area of polymers is higher than silica-based bonded phases, a smaller amount is used in cartridges, disks, and well plates. Thus, smaller amounts of sample and solvents as well as faster extraction times and faster evaporation times with reconstitution are required. 

Table II: Sample preparation products (continued) 

Products for QuEChERS were first introduced two years ago (2). Pronounced "catchers," QuEChERS is an acronym for quick, easy, cheap, effective, rugged, and safe sample preparation. This method is an emerging technique becoming increasingly popular in the area of multiresidue pesticide analysis in food and agricultural products. In QuEChERS, food and agricultural samples are extracted with first an aqueous miscible solvent (for example, acetonitrile) in the presence of high amounts of salts (for example, sodium chloride and magnesium sulfate) and buffering agents (for example, citrate) to induce liquid-phase separation and stabilize acid- and base-labile pesticides, respectively. Upon shaking and centrifugation, an aliquot of the organic phase is subjected to further clean up using SPE. Unlike traditional methods using SPE tubes, dispersive SPE cleanup is conducted by mixing bulk amounts of SPE sorbent and additional MgSO

 with the extract. After sample clean up, the mixture is centrifuged, and the resulting supernatant can either be analyzed directly or can be subjected to (minor) further treatment before analysis. Three new QuEChERS product offerings were submitted for inclusion into my Pittcon 2010 article. (see Table II). For those interested, a brief introduction to the technique was published in an earlier "Sample Preparation Perspectives (3). One aspect of this "just enough" sample preparation technique is that it is now extending beyond the analysis of pesticides in fruits and vegetables and has been used for additional classes of analytes in a diverse number of matrices (for example, antibiotics in meat and  PAHs in soil). 

In addition to QuEChERS, which is used primarily in food-safety analysis, other applications-specific SPE phases were introduced this year. In the environmental area, the SPE technique is now well accepted by regulatory agencies as a substitute for older methodologies like liquid–liquid extraction. New SPE products for EPA method 8270 for semivolatile organics in drinking and surface water were introduced at Pittcon. Other application-specific phases for oligonucleotides, trace organics in aqueous samples, acids and bases, drugs of abuse, proteins, drugs in plasma, and phospholipids in plasma were to be seen on the exhibition floor. 

One very interesting sample prep column was Varian's StratoSpheres PL-HCO3 MP. The large-volume cartridge is used to remove trifluoroacetic acid from HPLC effluents. Often, when proteins or peptides are purified by HPLC, they are isolated as their trifluoroacetic acid salt; the trifluoroacetic acid must be removed before further processing. This often is accomplished by repetitive freeze-drying, which is time consuming and costly. This quaternized polymer-based strong anion exchanger with a bicarbonate counterion can remove 1% trifluoroacetic acid  from 500 mL of solution by merely passing the liquid through the cartridge. 

Membranes are often very useful as sample preparation media. By selecting the appropriate molecular weight cut-off membrane, proteins can be excluded or alternatively, salts can be removed from protein mixtures. Figure 1 shows the desalting and concentration capabilities of a membrane-based filter. The Amicon Ultra-0.5 mL filter (Millipore, Billerica, Massachusetts) can remove salts or exchange buffers by concentrating the sample, then reconstituting the concentrate to the original sample volume with any desired solvent. The process of "washing out" can be repeated until the concentration of the contaminating microsolute has been reduced sufficiently. 

Chromatography and Sample Preparation Accessories

Table III lists 41 new hardware products and accessories for HPLC, GC, CE, and sample preparation. This year, I have extended the columns and sample preparation accessories to include small, benchtop-dedicated instruments that are designated to automate some of the sample preparation techniques reported in Table II. All of the products introduced were practical devices designed to make the chromatographer's life easier. I will cover each area individually. 

Everybody uses vials for automation; instruments are getting more sensitive. So this year, a new breed of ultra clean, GC–mass spectrometry (MS) and LC–MS-tested certified vials were introduced. In addition, a fused vial–insert combination designed to eliminate movement and potential needle damage was shown. The manual capping and decapping of vials takes a lot of time and can cause fatigue. To automatically cap and decap vials, a decapper system was introduced this year. New precut septa were shown. The slit in the laminated septum allows easy needle penetration yet prevents evaporation of volatiles from the vial. Finally, color-coded manual syringes help those mix-ups that can occur when quickly picking up a syringe to perform injections or liquid transfer. 

HPLC, LC–MS, and Ion Chromatography Products: 

With the current interest in UHPLC, peripherals capable of handling high pressures have begun to emerge. The UHPLC instruments now have pressure capabilities up to 1300 bar so system components need to stand up to these pressures. In addition, because UHPLC columns are very sensitive to particulate contamination, in-line filters designed to prevent plugging are becoming commonplace. This year, high-pressure fittings, in-line filters, on-line traps, and an injector were introduced. An example of one of the UHPLC peripherals is shown in Figure 2. The Optimize Technology (Oregon City, Oregon) EXP trap columns for sample cleanup can be used in a direct connect configuration via a holder (Figure 2a) or can be inserted into a six- or ten-port valve for column switching applications (Figure 2b). 

In the same vein, guard columns also offer an additional level of protection not only for UHPLC columns but conventional columns as well. Four companies introduced guard columns to meet this need. 

Other notable LC accessories introduced included a chiller/heater, fraction collector, a method development add-on instrument, a rack to hold LC modules, and an in-line bubble trap. 

 With the world's supply of helium dwindling and becoming more expensive, users are looking for other carrier gas sources that can provide an inexpensive, safe, pure, and plentiful gas supply. Hydrogen gas has superior properties to helium in terms of diffusivity and viscosity but users often are concerned about having additional cylinders of hydrogen around the laboratory. Hydrogen generators can provide carrier gas without fear of safety because only a few tens of milliliters are actually present at any given time. A new hydrogen generator was introduced this year. For performing quantitative analysis of volatiles, preparing standards is always a hassle. A new instrument designed to generate calibration standards automatically is a welcome addition to the GC laboratory. An improved instrument that performs end-to-end GC capillary column connections with no dead volume and that is capable of high-temperature operation without leaking was shown at Pittcon. 

Table III: Chromatography and sample preparation accessories 

Devices such as evaporators, concentrators, homogenizers, cryomills, and heater blocks are designed to take away some of the drudgery of sample preparation. In addition, small robotic devices automated part of the sample preparation workflow such as extraction and evaporation. All of these sample preparation products are always found on the Pittcon floor. Table III lists some of those products shown this year, many with improved features over previous models. 

Table III: Chromatography and sample preparation accessories (continued) 

Solid-phase microextraction (SPME) is a favored sample preparation technique mainly for GC. SPME fibers have a finite lifetime and must be changed periodically. For its MPS sampling system, Gerstel has developed an accessory in which the SPME fibers can be changed automatically at time intervals specified by the user. This feature improves overall laboratory throughput and productivity. 

I would like to thank the manufacturers and distributors who kindly furnished the requested information in advance of Pittcon 2010, thus allowing a timely report on new product introductions. Manufacturers who would like to be considered for inclusion into Pittcon 2011 coverage: please send primary company contact, mailing address, FAX and e-mail address to David Walsh, Editor, 

, c/o Advanstar Communications, 485 Rte. 1 South, Bldg. F, Iselin, NJ 08830, Attn.: Pittcon 2011 "Column Watch." 

"Column Watch" Editor Ronald E. Majors is Senior Scientist, Columns and Supplies Division, Agilent Technologies, Wilmington, Delaware, and is a member of LCGC's editorial advisory board. Direct correspondence about this column to "Column Watch," LCGC, Woodbridge Corporate Plaza, 485 Route 1 South, Building F, First Floor, Iselin, NJ 08830, e-mail 

Column Watch

Ron Majors presents the second part of his yearly review of all that was new and innovative at Pittcon 2010, held this year in Orlando, Florida.

New Chromatography Columns and Accessories at Pittcon 2010: Part II

Normally we use the calibration standards in a liquid chromatography (LC) method as a tool for quantitative analysis so that we can measure the concentration of an analyte in our samples. In this month's "LC Troubleshooting," we'll look at another use of the standards — to monitor the quality of the data, with a goal toward helping us in troubleshooting instrument problems. 

As an example, we'll use a high-precision method, such as might be encountered for a drug content method in the pharmaceutical industry or an active ingredient determination in a pesticide formulation. This kind of method is characterized by signal-to-noise ratios of >100 and peak area imprecision of ≤1% for several consecutive injections of the same sample. By examining a trend through a batch of samples or looking at individual outliers, we often can get enough information to help isolate the problem source to a particular module or submodule of the LC system. 

As with most problems, it is difficult to determine if something is wrong unless we know the normal behavior of the system. For example, let's consider the behavior of the calibration standards for a batch of samples. 

In this case, three different calibrators (STD A, B, and C) of nominally the same concentration were injected in duplicate followed by 6–10 samples in duplicate, then another set of standards, and so forth, finishing with a final set of standards. An example of the results for standards in a normal run is shown in Table I.  

Table I: Calibration data for a well-behaved method 

In Table I, we can see that the variability of peak area is very small, whether we examine the percent relative standard deviation (%RSD) for each calibrator over the entire run or compare the percent difference between consecutive injections. In all cases, the values are well below 1%. These data are typical for this method and will serve as our reference values for the problems discussed later. 

When the variability in peak area increases, the autosampler often is the first module to suspect. A good first step in troubleshooting is to make two or more injections from the same vial of the suspect sample or samples. If you normally inject duplicate calibrators from the same vial (a good idea), you will have this information as a normal part of the data set without having to go back and make special injections for troubleshooting purposes. If the variability in peak area is much larger than normal, this means that the same sample volume is not being injected each time, pointing to an autosampler problem. Check for obvious problems, such as a blocked or partially blocked autosampler needle, or insufficient sample. Once these sources are eliminated, look for mechanical problems with the autosampler.  

Many people erroneously assume that fluctuating area counts or response factors are associated with only the injection valve; however, there is another part of the autosampler in addition to the injection valve that contributes to the precision of the data and is subject to wear and tear, as well: the sampling unit, the part of the system that withdraws sample from the vial and transfers it to the sample loop.  

An example of problem data is shown in Table II. Here, a coworker's data shows mostly good agreement with area counts, with a few exceptions shown in bold italics. Notice that the occasional erratic peak areas throw off the %RSD values for the calibrator sets as well as the %-difference for the individual injection pairs. Understanding the functionality of the parts of the modules is crucial for troubleshooting. The injection valve directs flow to the column once the sampling unit has drawn the sample into the sample loop; therefore, if a cross-port leak was indeed the culprit, the data should show more variability than it does. The response would be less than normal as well, because a cross-port leak usually causes sample to leak into the waste stream. Hence, the injection valve was summarily dismissed as the problem, so we are left with the sampling unit as another potential problem source. Upon inspection of the instrument, it was discovered the metering drive, the part of the sampling unit that operates the sampling syringe, was loose. After tightening the bolts that hold the metering head in place, excellent precision was restored. 

Table II: Data set showing occasional abnormal injections 

In this example, the method normally gave very small %RSD values for standards, usually ≤0.5%. However, we noticed a trend in the data shown in Table III, with the overall %RSD about twice that normally observed (compare with Table I). Notice that, although each pair of consecutive standards agrees closely with each other, the trend is to larger variation through the run. Because each pair of injected standards was consistent (only STD A is shown here for brevity), we did not suspect initially that the autosampler was the problem. Again, in our experience, autosampler problems tend to show random variations in area, whereas in this case, there is a trend over the run. We suspected that this was due to increased noise from a detector lamp that was failing. 

Table III: Data showing higher-than-normal %RSD 

The condition of the lamp was confirmed by three different observations. First, according to the lamp life meter, the lamp had logged more than 1000 h. Lamps typically last between 1000 and 2000 h. If the lamp has logged >1000 hours and other symptoms exist, it is prudent to change the lamp. Second, this method uses a detector wavelength of 205 nm; lower wavelengths are the first to fail the intensity test performed on the lamp. This particular lamp barely passed the intensity test in the 200–220 nm range, so it was replaced. The final convincing argument for a bad lamp was the drop in %RSD from 0.80 to 0.21 when a new lamp was installed (Table IV). In addition, increased baseline noise is a common symptom of a failing lamp, but often this occurs only after the earlier symptoms are noted; increased baseline noise was not observed in this case. 

Table IV: Data from same system and method as Table III after detector lamp change 

To avoid premature lamp replacement after being misled by a drift in precision, as in Table III, it is important to allow the lamp to warm up for 30–45 min before analysis. A simple way to convince yourself of this requirement is to equilibrate the column for a method and load standards into the autosampler tray. Turn on the detector and immediately start injecting. Then compare the variability of early injections to those after the lamp is fully warmed up.  

An example of this is shown in Table V for a 15-min run. The column was equilibrated, and then the detector was turned on and injections were begun immediately. Although the %-difference in area between injections is abnormally high only for the first two injections, it can be seen that the overall %RSD improves if the first few injections are dropped. This is summarized at the bottom of Table V, where 

 = 12 is for all samples except the first one, and so forth. There is a noticeable reduction in the %RSD when the first one or two samples are dropped; after that, the value settles down, which would suggest that a 30-min detector warm-up would be sufficient. However, note that for maximum performance in this example, if 1.5 h elapsed before data were used (starting with injection 7), the %RSD drops to <20% of what it was with all the injections included. And yes, this is one data set on one detector, so specific warm-up time recommendations are not possible for all detectors and methods, but the pattern is consistent: detector performance improves if adequate warm-up time is allowed. 

Table V: Effect of lamp warm-up on peak area 

The last example is for the same high precision method, but an unusually high variation of areas was observed when comparing the early and late runs for the standards (see Table VI). Note that the areas for runs 45, 65, 66, and 67 (bold italics) are approximately 8% larger than the other runs. Because this instrument had been serviced recently and the precision for subsequent runs was acceptable, the autosampler was ruled out as the most likely cause of the problem. When the data were examined more closely, it was noted that the retention times also changed in conjunction with the area counts. A close examination of the system showed that the tubing between the aqueous solvent reservoir and the pump was filled with bluish particulate matter. This was microbial growth resulting from a phosphate buffer that was allowed to sit idle on the LC for an extended period of time without flushing with fresh water. The tubing was disconnected from the pump and hot water was forcefully flushed through the tubing, including the tubing inside the in-line degasser. This water was collected and filtered through a 0.45-μm PTFE filter to collect the residue. The result is shown in Figure 1, where the rinse water residue is compared with the residue from filtering an equal volume of HPLC-grade water. 

Table VI: Data obtained from contaminated LC system 

There are at least three possible consequences of this contamination. First, the frit in the mobile phase reservoir could be blocked. Second, the proportioning valve on the low-pressure mixing manifold could be malfunctioning. Third, the check valves in the pump could stick as a result of this fouling. Each of these three possible results was considered in light of the symptoms. A blocked reservoir frit would restrict flow of the aqueous solvent to the mixer. Because a constant flow of mobile phase is pumped from the mixer, when the organic solvent valve opens, excess organic solvent is delivered to make up the shortfall of the aqueous solvent. This means that the concentration of organic solvent in the mobile phase would be higher than normal. This is an ion chromatography method, where the buffer is the strong solvent, so higher concentrations of buffer would reduce retention times. Without sufficient buffer reaching the column, the analyte would be retained more, consistent with the observations. The inlet filter was changed, given the amount of microbial growth found in the low pressure lines. Finally, a partially blocked or intermittently sticking check valve would result in a lower flow rate than normal, and this would increase the retention time as with the Table VI data. A good practice is to note the stability of the pump pressure while the system is equilibrating and during system suitability. The pressure should be steady, varying no more than 1–2 bar (15–30 psi). If the pressure varies more than this, either there is a bubble in the pump that needs to be removed, additional mobile phase degassing is needed, a check valve is sticking or leaking, or some other flow-related problem is present. It isn't clear in retrospect which possible problem source was the root cause of the problem, or perhaps if a combination of causes existed. It also is not immediately obvious why the peak areas changed as much as was observed. In any event, flushing the system to remove residual particulate matter, replacement of the inlet frit in the reservoir, and sonication of the check valves resulted in a restoration of the LC system to satisfactory operation. 

This example drives home a point that has been mentioned in this "LC Troubleshooting" column regularly over the last 25 years:  Do not turn off the LC system with buffer in it! Sure, a few minutes won't hurt, but extended standing with buffers encourages evaporation of the liquid phase and formation of buffer crystals as well as microbial growth inside the system. A good habit is to replace the buffer once a week, although many laboratories will use buffer for two weeks without problems. And before you shut down the system, flush all the buffer lines with water. It is best never to reuse a buffer reservoir — use all the buffer, then replace the reservoir with a clean one. A used reservoir can inoculate the next batch of buffer with microorganisms contained in the last batch. If you find yourself in possession of a contaminated system, as was the case here, replacement of the tubing would be a good idea, and flushing with a dilute solution of chlorine bleach also would ensure that any microbial contaminants had been deactivated (obviously, you shouldn't pump bleach through the column!).  

The examples cited this month illustrate several problems that were identified initially by studying the behavior of calibration standards after a sample batch was completed. In addition to calibration properties, these standards can serve as a powerful indicator of the health of the LC system. In each case, the symptoms pointed to a general part of the LC system. Further observation and interpretation of the data helped to isolate the problems so that they could be corrected. 

Jennifer Birchett is an analytical chemist with Syngenta Crop Protection, Inc., Wilmington, Delaware. She is responsible for the development and validation of methods for herbicide products as well as the on-demand troubleshooting and repair of 30 departmental LC systems.

"LC Troubleshooting" Editor John W. Dolan is Vice-President of LC Resources, Walnut Creek, California; and a member of 

's editorial advisory board. Direct correspondence about this column to "LC Troubleshooting," 

, Woodbridge Corporate Plaza, 485 Route 1 South, Building F, First Floor, Iselin, NJ 08830, e-mail 

For an ongoing discussion of LC troubleshooting with John Dolan and other chromatographers, visit the Chromatography Forum discussion group at 

LC Troubleshooting

In this month's "LC Troubleshooting," the authors look at another use of calibration standards: to monitor the quality of the data, with a goal toward helping scientists troubleshoot instrument problems.

Getting the Most Out of Calibration Standards

Issue PDF

Click the title above to open the LCGC North America April 2010 regular issue, Vol 28 No 4, in an interactive PDF format.

LCGC North America-04-01-2010

Could This Be Your MS System of the Future?

Simultaneous Determination of Synthetic Pharmaceutical Peptides and the Acetate Counterion by Mixed-Mode Weak-Anion-Exchange Chromatography

New Chromatography Columns and Accessories at Pittcon 2010: Part II

Getting the Most Out of Calibration Standards

Vol 28 No 4 LCGC North America April 2010 Regular Issue PDF