In early 2007, a web-based HPLC columns survey was conducted. Current usage was compared to a 1997 survey. Some interesting trends were observed. Although reversed-phase chromatography maintained its dominance, techniques that grew in importance were chiral, HILIC, and biochromatography applications. Smaller 3.0-mm particles have gained in popularity while sub-2-mm columns are now getting attention. Column life has increased with guard columns being a major factor. Column buying trends were analyzed: average number of columns used per instrument per year remained at 5; price increases have stayed at the rate of inflation; a major shift in purchasing columns from general laboratory distributors. Column-to-column reproducibility is still the top factor in buying decisions. Future column interests include hybrids, sub-2-mm, and ultrahigh-pressure columns

Occasionally, LCGC surveys its readers to obtain a current profile of users of high performance liquid chromatography (HPLC). The last general survey on HPLC columns was conducted was in 1997 (1). In the past, I have used results from these surveys to chart trends in column technology and the practice of HPLC. In early 2007, a web-based survey was conducted and 221 respondents answered a series of questions on their mode and column usage. Although the number of respondents was reduced from earlier surveys (2–7), the number of respondents was statistically sufficient to allow comparisons to previous survey data. 

To ensure that the results of the current survey were compatible with those of previous surveys, I used the same methodology to report the results. Because many of the questions allowed respondents to give more that one answer, in some cases, I normalized response totals. Normalizing the results to a base of 100% makes it easier to compare the results of previous surveys with those of the present survey and to identify trends in the use of HPLC columns, modes, and packings. Questions pertaining to mode usage, column life, particle sizes, purchasing considerations, and possible future needs were explored to understand selection criteria. 

To understand the current usage rate of instruments, a question was asked pertaining to the number and type of instruments used per respondent. Table I shows that the average response for each category of instrument. The numbers reported should not be construed that every respondent uses every type of instrument but serves to provide an idea of the relative number of the various types of systems in use. Later, I will look at the numbers of columns used by these instruments.  

Table I: Types of HPLC instruments used by survey respondents			

According to the survey results, a conventional HPLC instrument user is responsible for an average of 2.9 instruments. This number was derived from looking at the total number of instruments of the type identified by all respondents and then dividing this number of the total number of respondents. For example, some respondents reported that they have only one conventional HPLC system while others reported that they have four instruments for which they are responsible. Based upon the relative numbers of Table I for every one of these respondents that uses a conventional LC system, only 1 in 11 would possess a capillary or nanoLC system, 1 in 5 might have an instrument capable of working with microbore columns and 1 in 7 could have a preparative instrument in his or her laboratory.  

Most liquid chromatographers use more than one chromatographic mode to solve specific separation problems. The total number of techniques used by respondents relative to the number of respondents revealed that each used 2.5 modes on the average, slightly less than the results from the last survey (1). The normalized data in Table II compares the relative mode usage in 1997 and 2007. No surprise that the results again show that reversed-phase chromatography was the most popular mode; according to the raw data, over 92% of respondents use this mode with normal bonded phases a distant second.  

Table II: Analytical HPLC mode and stationary phase usage			

Although the data in Table II shows that reversed-phase chromatography has dropped on a relative basis, this drop is not due to the decrease in popularity of this mode but rather from the increased use of other modes such as chiral chromatography, hydrophilic interaction chromatography (HILIC) and those modes popular for biomolecules (such as ion exchange, hydrophobic interaction and affinity chromatography). In fact, in 1997, HILIC did not even make the charts as it was relegated to a few peptide applications. HILIC is a separation technique for highly polar analytes that gets around some of the problems associated with reversed-phase chromatography such as low retention or phase collapse (dewetting). HILIC uses a polar stationary phase such as bare silica gel or polar bonded phase (for example, diol) and requires a high percentage of a nonpolar mobile phase, similar to the requirements for normal phase chromatography. However, unlike normal phase, which uses nonpolar solvents like hexane and methylene chloride and tries to exclude water from the mobile phase, HILIC requires some water in the mobile phase to maintain a stagnant enriched water layer on the packing surface into which analytes might selectively partition. In addition, water-miscible organic solvents are used. Under HILIC, polar analytes are well retained and are eluted in order of increasing hydrophilicity.  

With regulatory bodies requiring evidence of enantiomeric purity, it is no surprise that use of chiral chromatography has increased by 60% in the last decade. Newer chiral columns appear to be more universal and some can be used in both the reversed-phase and polar organic modes. Supercritical fluid chromatography (SFC) also has seen renewed interest, especially for preparative separations. Columns applied to SFC are similar to those used in HPLC.  

The use of normal bonded phases continues to see widespread application. This mode is favored over adsorption chromatography for the separation of geometric and positional isomers and 

 compounds and other separations using organic mobile phases where reversed-phase chromatography does not do a good job. The phases used in this mode such as diol, amino, and especially cyano are sometimes used with aqueous solvents. The survey did not solicit information about the uses of these stationary phases in aqueous versus organic mobile phases.  

The use of ion-exchange chromatography showed a slight upward swing in usage and is back to its historical levels of the mid-to-late 1980s (1). Included in this usage is ion chromatography that is sometimes treated as a separate separation technique but in reality is ion-exchange chromatography because it employs the same separation principles and uses columns with ionic or ionizable functionalities. The increasing popularity of ion exchange can be somewhat attributed to the strong interest in proteomics where large number of tryptic peptides are fractionated by ion exchange before reversed-phase chromatography–MS separation and detection. Although ion-pair chromatography also separates ionic and ionizable compounds, it is generally lumped with reversed-phase chromatography because it employs the same packing materials. Many chromatographers familiar with reversed-phase chromatography prefer to use ion-pair chromatography for ionic and ionizable compounds because the columns are more familiar to them and the principles of reversed-phase chromatography can be generally applied in method development. 

The data in Table III breaks the reversed phases into individual organic moieties and clearly shows that C18 (octadecylsilane) was most popular (the raw data showed that 92% of all users use this phase at some time) followed by C8 (octylsilane). The use of phenyl bonded phase has increased as users exploit its unique selectivity compared to alkyl phases. In recent years, the introduction of alkyl chains greater than C18, up to C30, has occurred. These long chain phases provide a greater degree of shape selectivity than a typical C18, have long proven superior for the separation of 

 carotenoid isomers, are resistant to phase collapse at low percentage of organic solvent in the mobile phase and have high loadability.  

Table III: Stationary phases used in reversed-phase chromatography			

In the current survey, the use of polymeric packings was examined. Only 13% of the respondents indicated that they were using polymeric columns for their HPLC work, down from 17% in 1997 (1). This is somewhat surprising because polymeric packings have improved and current column efficiencies have narrowed the gap compared to silica-based packings of the same particle size. Of those using polymeric columns, the mode breakdown is reversed phase (77%), ion exchange–ion chromatography (47%), and size-exclusion chromatography (50%), equally split between nonaqueous (gel permeation) and aqueous (gel filtration). The most popular non–size exclusion applications for polymeric columns are high pH (where traditional silica gel–based columns have solubilization problems), organic acids, proteins–peptides, inorganic ions, carbohydrates, and amino acids.  

An open-ended question pertaining to the use of chiral columns in analytical HPLC revealed that columns based upon polysaccharide chemistry dominate the applications but macrocyclic antibiotic, protein-based (for example, α-glycoprotein), and Pirkle-type columns do command attention in certain application areas.  

Particle Sizes in Analytical and Preparative LC

Since the beginning of HPLC in the early 1970s, the trend has been to use columns packed with smaller spherical particles. Although irregular microparticulate particles were the first to be introduced (8), they quickly gave way to spherical particles that could be packed more reproducibly, gave slightly lower pressure drop, and provided column efficiency needed at the time. For cost reasons, irregular particles are still sometimes employed in preparative chromatography but advantages of the spherical particles when reclaiming packing from packed beds is evident.  

In the 1970s, 10-μm irregular particles were the norm because they were available commercially and with slurry packing column efficiency was quite good. However, studies had shown (9) that smaller particles gave better performance due to improved mass transfer but these smaller particles were not freely available until the early 1980s. Through occasional 

 column surveys, I started tracking the use of particle sizes in the mid-1980s. Table IV provides data of the use of various particle sizes from 1985 until 2007 (current survey). By 1985, columns packed with 10-μm particles were already giving way to 5-μm particles that maintained their dominance through the 1990s and are still in widespread use today. There are many validated methods that have been developed on 5-μm particles and users do not want to take the time to revalidate their method unless there is a strong driving force to do so. Also, for many applications, a 25-cm column provides a sufficient number of theoretical plates to provide adequate resolution. The use of even smaller particles (3–4 μm) began in earnest in the mid-1990s and now, as indicated in Table IV, these particles have become widely used. With the reduction in particle size, column efficiency improved further allowing a reduction in column length. Shorter columns are now in vogue. Not only does a shorter column provide faster separations but also solvent use is reduced. Recently, the use of sub-2-μm particles has become fashionable in even shorter column lengths (10). Columns of 20–50 mm in length now provide plate counts formerly obtained on 15- and 25-cm columns packed with larger particles. In the next survey, it will be interesting to see if the trend on the increased use of small particles continues. 

Table IV: Analytical HPLC particle size trends 1985â2007*			

In comparing the use of preparative chromatography to analytical HPLC, about 1 in 4 respondents who do analytical work also use preparative chromatography. Not surprisingly, those that perform preparative separations tend to prefer larger particles as depicted in Table V. Some of the reasons for the preference of larger particles is cost, pressure drop at high flow rates and loadability. For the latter consideration, the higher efficiency of smaller particles can be lost when overloaded conditions are employed. 

Table V:  Particle sizes used in preparative chromatography			

Several questions in the survey related to the purchase and cost of analytical HPLC columns on a per-instrument basis. Most of the respondents purchase prepacked columns but a few purchase empty columns and bulk packings presumably to pack their own; still, a majority of respondents have no budget for empty or bulk packings. Another question later in the survey revealed that only 0.5% pack their own columns. After prepacked columns, the largest expenditure is for guard columns. One question was asked on how their budgets are allocated among prepacked columns, empty columns, column packings, and guard columns. The averages of all responses were 59%, 5.3%, 9.3%, and 26%, respectively, not that much different than 10 years ago (1). Based upon the question, about 73% of the chromatographers using prepacked columns also purchase guard columns.  

The average annual purchase budget for prepacked analytical HPLC columns per instrument was approximately $2950, about 30% higher than the survey results of 10 years ago (1). Based upon a unit cost of $400, this amount would be equivalent to just over seven columns a year per instrument. Guard column budgets were about $1300 per year per instrument. For the average cost of a guard column of $80, this would amount to approximately 16 guard columns per year, not an unreasonable number. Again, this expenditure was just less than 30% higher than the 1997 survey (1). Guard columns extend the life of analytical columns; therefore, without these expenditures, users would have to allocate a higher percentage of their budget for prepacked columns. 

Another question broke down the purchases of different dimensions of LC columns. Users were asked about their yearly purchases of conventional analytical columns (3.0–4.6 mm i.d.), microbore columns (2.0 mm i.d.), narrowbore columns (1.0 mm), and capillary columns (less than 1-mm i.d.). Table VI shows the column units used based upon a weighted average. For conventional HPLC column users (3.0–4.6 mm i.d.), an average of five columns per instrument per year are used. Even though the previous results showed that the respondents purchased seven columns per instrument per year, this number was based upon an average cost of $400 per column. Some columns such as chiral columns and size exclusion columns might cost double or triple that number so it is not surprising that there was a slight discrepancy in the purchase versus use numbers. In Table VI, other column types also are shown. The short, fast LC category could just as well fall into the conventional analytical sizes because many of these popular columns have 4.6-mm internal diameters. One would expect narrow bore and capillary columns to have lower usage based upon the smaller number of available instruments that accommodate these smaller internal diameter columns (see Table I).  

Table VI: Types of analytical columns used by espondents			

A question asked if the average price of an HPLC column had changed in the last three years and 58% of the respondents felt that column prices had stayed the same while 35% felt that prices had increased. The average perceived price increase for the latter group was 60% or about 16% a year increase. In reality, judging from the column budgets discussed previously, the increase in column prices per year over a 10-year window was less than 3% per year. Only 4% felt that column prices had decreased in the last three years. 

One of the questions asked chromatographers from which source they prefer to purchase prepacked HPLC columns. According to the survey, Table VII shows that, compared with 1997 (1), the chromatography supply houses maintained their lead but their market share dropped since 1997. The biggest growth was for the general laboratory suppliers; in 1994 (7) they were a mere 7% of the market growing to 11.3% in 1997 (1). This year 30% of the users surveyed purchase their columns from this source. General laboratory distributors usually provide less technical assistance that the dedicated chromatography suppliers or instrument manufacturers but they promote themselves as one-stop shopping where users can get solvents, chemicals, laboratory glassware as well as HPLC columns. The general laboratory distributors sometimes provide additional services such as electronic ordering, local stocking, volume discounts, and on-site services. According to the present survey, the biggest decrease in column purchases was from the independent column manufacturers who saw a substantial drop in the share of the HPLC columns' market.  

Table VII: Sources for HPLC column procurement			

Table VIII lists the factors that respondents considered important when purchasing an HPLC column. Respondents were asked to name their top three factors that influenced their buying decision. The most important factor — ranked the same in earlier surveys — is column-to-column reproducibility. Nobody wants to develop a method only to find out that the column that they purchase later for the same application does not perform in the same manner. However, column reproducibility has definitely improved over the years driven by competitive pressure, quality initiatives such as ISO and 6-Sigma, and a better understanding of the manufacturing processes. The next three top factors were the same as in the 1997 survey (1) but their relative importance has changed. For example, column life has increased in its relative importance. Nowadays, users expect more injections per column than they did a decade ago and column life affects their buying decision. (Note: we will explore this factor in more detail in the next section). This year I included tailing factor which seems to be quite important in the pharmaceutical laboratory that deals with basic drugs that have a tendency to tail on some silica-based bonded phase columns.

Table VIII: Factors considered when selecting an HPLC column supplier			

As mentioned earlier, column life is an important selection and operating parameter in HPLC. This survey specifically explored column life from two standpoints — lifetime in months and by the number of injections. Although some of the modern liquid chromatographs do track the number of injections, most chromatographers do not. Usually, one will install an HPLC column and keep it in the instrument performing one or more methods until the column shows a loss of resolution or shows significant tailing that might affect quantitation. They can track the months of usage in a logbook.  

Table IX: Average analytical column lifetime			

As can be observed in Table IX, since the earliest days column life continually has increased through the years. I first began asking about life in column surveys in 1984 and for a majority of respondents, columns lasted less than three months. Now only 15% of respondents have lifetimes of less than three months. If one looks carefully at the data of Table IX, nearly 20% of the respondents now experience lifetimes of at least a year and over 30% have columns that last over a year, higher than data reported in the 90s. About 20% of the users do not track column life at all.  

Table X: Normalized percentage of respondents who reported on column life by the number of sample injections for analytical columns			

When the question was asked about column life based upon the number of injections, a larger number (31%) of the respondents reported that they do not track their injections. Table X shows the normalized data of those who do monitor column life based upon the number of injections. Compared to Table IX, the results are not as clear in terms of the 1997 data and the 2007 data. Clearly, a significant larger number of respondents (37%) in 2007 indicated that they can achieve more than 1000 injections compared with 1997. However, some of the other levels of injection tracking have shown a decrease in the number of injections in the more recent study. When looking at those achieving 500 or more injections, there was a definite increase in the 2007 study. 

Table XI: Number of sample injections before replacing guard column			

Although the data is not shown, a large percentage of preparative column users do not track column life nor the number of total samples injected before discarding a column. Intuitively, one would think that preparative columns would have shorter lives because they tend to be used for dirty samples, at high flow rates, and higher sample loads than analytical columns. Surprisingly, respondents who use preparative columns reported that nearly 50% of them last longer than a year, similar to the results reported in 1997 (1). This greater life perhaps indicates that preparative columns are used less frequently than analytical columns and therefore tend to be stored and used at a later date when needed for preparative applications. Another possibility for longer lives would be that preparative columns are substantially more expensive than analytical columns and users might take better care of them. A third possibility is that the larger particles used in prep do not generate as high a backpressure as obtained with the smaller particle analytical columns. Higher backpressure tends to limit the column life because more stress is placed on the column materials. 

Everybody is interested in reducing the operating costs in their laboratories. Approaches to reducing the costs of columns, supplies, solvents and other chemicals are of increasing importance for everyone from bench chemists to laboratory managers to purchasing agents. As was mentioned earlier, column lives have increased over the years. This life increase should imply that fewer columns need to be purchased per year. The increased life of columns is due to a number of factors: better manufacturing procedures; a better understanding of users on the limitations of bonded phase silica columns; newer bonded phases that withstand low and high pH; newer types of higher stability phases such as zirconia, silica–organic hybrids; and increased use of guard columns and in-line filters. 

The use of guard columns has helped to decrease the cost of replacing the more expensive analytical columns. A question was asked about the frequency of change of guard columns. For those that use guard columns (73% of respondents), Table XI shows the results of the guard column usage survey with comparative data from 1997 (1). From the data, the number of respondents using guard column has increased since the 1997 survey. In addition, it can be deduced that guard columns are also lasting longer than a decade ago. For example, the percentage of respondents who get more than 250 injections from guard columns increased by 75%. Undoubtedly, the end result is an increase in the life of the analytical column also. 			

The use of polymeric columns is another potential savings. Polymeric columns have a wide range of pH stability (see earlier comments) and are especially useful at high pH values where traditional silica gel bonded columns have stability problems. Even so, there has been a slight decrease in the use of polymeric columns in this present study. Newer bidentate silica and hybrid silica–organic columns have allowed higher pH operation and might have taken some market share away in the reversed-phase mode.  

The survey asked a question regarding future column interests. Table XII indicates that there is considerable interest in the newer hybrid columns that consist of both inorganic and organic character. These columns have the structural rigidity of the inorganic portion and the chemical stability of the organic portion. The sub-2-μm columns have struck the fancy of those who wish to decrease their analysis times by shortening the column length and to those who want to have greater plate count by using longer columns, albeit at the higher pressure. The third most popular topic for future consideration are those that will withstand higher pressures exceeding 1000 bar (~15,000 psi). Presumably, if the decrease in particle sizes for analytical separations continues such columns will become mainstream.  

Articles

Column Watch Column

In early 2007, a web-based HPLC columns survey was conducted. Current usage was compared to a 1997 survey.

Current Trends in HPLC Column Usage

A recurring topic in this column is structural characterization including the underpinnings of mass accuracy and resolution and spectral interpretation (1–3). You will find this month's column the practical culmination of many of these previous discussions. The need to determine the exact structure, especially of an unknown, is at the core of why we employ mass spectrometers in all the variations and "flavors." Mass spectrometry (MS) has developed into a mainstream analytical technique in the pharmaceutical industry where one of its major roles is identifying and characterizing new chemical entities by elucidating the structure of unknown compounds.  

Jean-Claude Wolff, a primary contributor to this discussion as a scientist with GSK (Medicines Research Centre, Stevenage, Hertfordshire, UK) has found characterizing unknowns in potential counterfeit medicines both a challenging and excellent focus for his talents.  

I would not suggest that elucidating the structure of impurities is easy. My purpose is merely to show how combined mass spectrometric techniques can gather the information necessary to elucidate the structure of an unknown compound. In so doing, I suggest a pathway that leads to identifying the structure of the molecule analyzed. As an example, I offer the exercise of determining the substitute, active pharmaceutical ingredients in counterfeit medicines.

Often the only information available when considering a novel chemical entity might be that the new, unknown compound of interest belongs to a particular class of compounds — in natural product research alkaloids, for instance. Additional information ("background" information) becomes available after performing structure elucidation on impurities or degradants structurally related to a pharmaceutically active compound.  

The World Health Organization (WHO) defines counterfeit medicines as: 

[T]hose medicines that are deliberately and fraudulently mislabeled with respect to identity and/or source. Counterfeiting can apply to both branded and generic products. Counterfeit products may include products with the correct ingredients or with the wrong ingredients, without active ingredients, with insufficient active ingredient, or with fake packaging [4].

"I focus on products with the wrong active pharmaceutical ingredient whose identity I then determine using MS techniques. My sole guidance is that a compound [to be a likely suspect] be readily available commercially and that it be cheap." These are the key elements making the compound attractive to the counterfeiters. Note, however, that "substitute actives" are not necessarily so common that they will be found in libraries. Hence, the reason for structure elucidation.  

Counterfeits with an incorrect active ingredient are commonplace. A study of antibiotics and antimalarials by the German Pharma Health Fund showed about 16% of cases investigated contained incorrect ingredients (5). 

The MS tools available to determine incorrect pharmaceutical ingredients are numerous: you can use a series of sample introduction techniques like direct inlet or probe, gas chromatography (GC) and liquid chromatography (LC). The choice of inlet technique depends on a sample's characteristics. Inlet technique is also linked to the ionization method you choose — electron ionization (EI), chemical ionization (CI), atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI) — or the newer techniques like desorption electrospray ionization (DESI) or direct analysis in real time (DART). All of these techniques have been reported in this column in recent years — though admittedly we treated GC techniques rather lightly, an imbalance we shall soon redress. 

Structural elucidation of unknowns (in this case substitute, active pharmaceutical ingredients) demands using MS instruments capable of tandem MS and accurate mass measurements to determine the elemental composition of an analyte. Here the choice is large, ranging from ion trap to Fourier-transform (FT) ion cyclotron resonance via quadrupole time of flight (Q-TOF). Regardless of the instrument or ionization technique used, however, you can elucidate an unknown compound's structure by using the arsenal of MS techniques available. The goal here is simply to show a possible pathway and discuss its merits. 

Structure elucidation of a substitute, active pharmaceutical ingredient in an antimalarial tablet: 

The most effective approach for identifying and characterizing counterfeit medicines is to analyze a suspect sample using LC coupled to accurate-mass, tandem MS. This approach is more precise and quantitative than direct-inlet analysis (probe or infusion) of a sample. LC provides distinct separation of the active or substitute-active pharmaceutical ingredient from the excipients. Moreover, using LC reduces the potential for ion suppression in the ion source. Another advantage of introducing a sample via LC is the ability to compare LC–UV and LC–MS profiles of a suspect sample with a genuine one.  

Wolff's preferred approach to minimize analysis time employs:  

a generic, 10-minute LC-gradient separation with a conventional C18, reversed-phase column, 1.8-μm particle size. The eluents are 0.05 M ammonium acetate in water and acetonitrile. This method works for most samples analyzed. Obviously, however, for particular samples, it would need changing. Even gas chromatography (GC) could be used, as GC–MS (with EI ionization) allows comparing the spectrum you obtain from a suspect sample to those of known compounds. However, a considerable number of samples are antibiotics (penicillins and cephalosporins), which are not easily analyzed by GC–MS. And so, on balance, I find that the LC–MS setup using positive-ion electrospray ionization permits me to analyze the vast majority of samples encountered.

Figure 1 shows the LC–MS chromatogram obtained when analyzing a suspected counterfeit antimalarial. The correct active pharmaceutical ingredient has a nominal mass of 499, so it should give an [M+H]

  peak at 500. The major peak in the LC–MS chromatogram, however, shows an [M+H]

  at 152. That the acetonitrile adduct ion appears at 

 152 represents the protonated molecule. The chromatogram offers no evidence for the presence of 

 500. Indeed, it shows only one major chromatographic peak. At retention time of 2.8 min, that peak is likely the substitute, active pharmaceutical ingredient. 

"I performed the analysis on a quadrupole, time-of-flight, tandem mass spectrometer (Qq-ToF) capable of accurate mass measurement. The next task was determining the elemental composition for the substitute, active ingredient — in this case for the peak at 

 152." Most instrument software calculates possible elemental compositions, possibly using all the elements of the periodic table. In practice, however, you would set limits on the elements you would consider. You can exclude some of them because they are rarely encountered in pharmaceutical and/or organic molecules. You can exclude others on the basis of the isotope pattern observed for the protonated molecule at 

 152. For instance, most metals have multiple isotopes, and you would observe an isotope envelope in the mass spectrum of the substitute, active pharmaceutical. The same applies to some halogens: for example, Cl and Br, which are frequently encountered in pharmaceutical molecules. These halogens evidence a characteristic isotope pattern: for example, Br isotopes are 

 Br and have a ratio of about 1:1. Likewise, the isotopic ratios of chlorine and sulfur are, respectively, 

As discussed in previous columns, how the accurate mass of an ion in a mass spectrum is measured is very important. The better the accuracy, the fewer the possible elemental compositions. Figure 2 shows (we have used this one in previous discussions in this column) the number of elemental compositions possible with a mass accuracy of between 1 ppm and 5 ppm when considering only the elements of carbon, hydrogen, nitrogen and oxygen (C, H, N, O). Obviously, the number of possibilities increases exponentially with mass. 

Setting realistic boundaries when calculating elemental compositions is crucial. If you set the limits too tightly, you can miss the correct elemental composition; setting the limits too loosly leads to the Herculean task of determining the correct elemental composition. You must therefore know your instrument's capabilities well. Employing the restrictions dictated by Figure 2, using a medium resolution typical of Q-TOF instruments (7000–17000 FHHW), a limit of 2 mDa could be sufficient if the measurement were made in conditions conducive to "optimal" operating conditions for good mass accuracy. (Note: optimal conditions would be correct ion abundance, adequate calibration of the instrument, and the use of an internal reference to perform the mass measurement.) For higher resolution instruments, such as FT, tighter limits of 0.5 mDa could be reasonable. 

To illustrate, consider the example where the nominal mass of a substitute, active pharmaceutical ingredient is 151, and the protonated molecule was mass measured to 152.0679. According to Figure 2, assigning the proper identity should be a relatively easy task because the number of elemental compositions possible for mass 152 should be limited. Elements considered for the elemental composition of the unknown are C, H, N, and O, as well as F and S, all of which are frequently encountered in pharmaceuticals. From a quick analysis of the isotope pattern, we know that there is no Cl or Br present. For F, the number of elements is limited to three and for S to one. 

Wolff's approach is "to be conservative — that is, to be certain to avoid missing the correct elemental composition, I use a 5-mDa accuracy limit, especially since I often cannot optimize the experimental conditions for best mass measurement (that is, possibly acquiring too high ion abundance). The number of possible elemental compositions for 152.0679 setting a 5-mDa tolerance and considering C, H, N, O, F(3), and S is six. If you increase the tolerance to 10 mDa, the number of possibilities increases to eight [Table I]. Only even electron elemental compositions are considered."  

Table I: Calculated elemental compositions for m/z 152.0679 observed in counterfeit tablet			

Electrospray ionization produces, almost exclusively, either protonated ions or cations (in the latter case elements like sodium and potassium need be considered). For other ionization techniques, like photoionization, it would be wise to consider both even and odd-electron elemental compositions. 

How do you pick the correct elemental composition from this list? Well, you could search for the elemental compositions in the 

 or Chemical Abstracts Service (CAS) to determine a correspondence to a readily available commercial compound. By doing so, you would get a hit for C

, an elemental composition that corresponds to acetaminophen (paracetamol). However, such an approach would be extremely tedious for higher molecular-weight compounds, returning a vast number of elemental compositions and as Jean-Claude pointed out the counterfeiters do not play by any rules other than "use the cheapest most readily available substitute" so it is wise to be circumspect. 

A more scientific and systematic approach consists at looking closer at the isotope pattern in the mass spectrum. This topic has been described in a recent column that focused on the work of Kind and Fiehn (3). Careful scrutiny of the isotope pattern of the ions measured lets you narrow the number of possibilities. Observing the 

C ratio can reveal the number of carbon atoms in an analyte. Naturally occurring carbon contains approximately 1.1% of the 

C isotope. So an ion containing 10 carbon atoms would display a 

C isotope peak with an abundance of 11% of the 

C peak. Similarly, an ion containing 50 carbon atoms would display a 

C isotope peak with an abundance of 55% of the 

C peak. A simple ratio measurement, therefore, gives a good indication of the number of carbon atoms present, within the precision of the mass spectrometer, which is typically up to about 15% (uncertainty can be larger due to the peak-centering process). 

In the case currently in question Wolff points out "the peak at 

C contribution, has an abundance of about 10% of the 

C peak), suggesting about nine C atoms. However, you must allow for some uncertainty because of possible skewing of the isotope pattern, the result of chemical background noise, or the centroid data format. In the end, therefore, you would consider elemental compositions with 8–10 C atoms."  

In Table I, only one elemental composition falls within the 8–10 C atoms range: C

, which corresponds to acetaminophen. "You can show that this is the case by plotting the elemental composition of the sample and comparing it with the theoretical isotope pattern of C

. Moreover, the isotope pattern of the peak obtained from the sample does not evince the presence of a sulfur atom. In the latter case, the 

 154 would be about 4% of the abundance of 

 152. However, for higher molecular-weight compounds, it can be more difficult to spot 

  peak also contains the contribution of two 

Analyzing the isotope pattern of the peak led to identifying the substitute, active pharmaceutical ingredient in this counterfeit tablet, namely acetaminophen. To confirm the identity, Wolff said, "I analyze a standard and match the MS spectrum and retention times. For more confidence, or in more challenging cases (higher molecular-weight unknowns), I would register a tandem MS spectrum and measure accurate mass to determine the elemental composition of the fragment (product) ions. Doing so is easier because, at lower mass (or 

), fewer combinations exist. In the case discussed so far, fragmentation of 

 110. In a first instance, since I am dealing with ESI, a soft ionization technique, only even-electron elemental compositions are candidates for the product ions. This means that the fragmentation mechanism is essentially based on neutral losses (loss of a neutral molecule), the most common in ESI tandem MS. But if no satisfactory even-electron elemental composition fits the measured accurate mass, you should envisage odd-electron elemental composition. Several papers in the literature describe odd-electron product ion formation [6]. In my experience, it arises frequently in electron-rich structures, in molecules that contain more than one S atom, and in molecules that potentially form very stable odd-electron (product) ions." 

 110, only five possible even-electron elemental compositions occur, even with a very conservative 10-mDa mass-accuracy limit (Table II). 

Table II: Calculated even-electron elemental compositions for product ion m/z 110.0579			

Only one of the five elemental compositions seems likely from a chemical point of view: C

NO. No mass spectrometrist or chemist would believe CH

 might be a prospective structure. Those have a double-bond equivalence (DBE) of –0.5, which means they are linear molecules (no ring, no double bond). The double-bond equivalence (calculated based on the valence of the individual atoms–elements in the molecule) can be a useful parameter to apply when narrowing possible elemental compositions. If a product ion, whose elemental composition is determined, has a double bond equivalence of 3.5, as in the present case, the precursor ion has a similar or higher DBE. It can be slightly lower (1–2 units) when, through the neutral loss, a double-bond or ring structure is introduced in the product ion. Absent that, however, you could set the minimum DBE to the value found in the product ion (for an initial approach). In the case of acetaminophen the resulting spectrum would leave only two elemental compositions for the protonated molecule 

F. Having thus far limited the outcomes would leave the molecules that give the closest isotope pattern fit. 

Wolff's assessment indicates, "The product ion for 

O — that is, acetyl — which is a frequent neutral loss in ESI tandem mass spectrometry. The relation between product ions themselves and the precursor ion can help build the unknown molecule from product ions whose elemental composition was determined, a method commonly used to identify structurally related compounds. A known compound, like the active pharmaceutical ingredient, is taken as reference MS-MS spectrum, and the spectra from the impurities are compared to the reference to determine what modifications occurred and where they occurred. If a product ion shows a +14 mass difference when compared to the reference spectrum, that part of the molecule most likely contains an extra –CH

Knowing the elemental composition of product ions can also set a minimum for certain elements in the elemental composition calculations. In the case of acetaminophen, the 

 110 product ion would set a minimum of one N atom and one O atom to be present in the precursor. This actually would eliminate C

F from the original list deduced above and even when discriminating based on DBE. So applying DBE and the minimum-number-of-heteroatoms (N and O) criteria would leave only one elemental composition: C

, the correct elemental composition for protonated acetaminophen. 

Looking at the parity of the product ions in the MS-MS spectrum could be useful in determining the correct elemental composition and ultimately the structure of a substitute, active pharmaceutical ingredient. Because of the nitrogen rule, an even-number protonated molecule contains an odd number of N atoms, and an odd-number protonated molecule contains no N atoms or an even number. So if in the MS-MS of an odd number protonated molecule even-number and odd-number product ions exist, it is likely that the molecule contains at least two N atoms (the hypothesis being that all product ions are due to neutral losses). 

Last, but not least, you might find it useful to decrease the resolution on the mass filtering quadrupole (quadrupole 1 on a triple quadrupole instrument or Qq ToF instrument) to select the whole isotope cluster of the precursor ion and, hence, record the full isotope pattern on the product ions. Doing so obviously helps assign elemental composition to the product ions as described earlier for the precursor ion. 

In the case discussed so far, all approaches led to identifying the substitute, active pharmaceutical ingredient in the counterfeit tablets as acetaminophen, a finding confirmed by recording an MS-MS spectrum for an acetaminophen standard and working out the fragmentation pathway observed (Figure 3). 

Structure elucidation of a substitute, active pharmaceutical ingredient in a degraded, reconstituted, injectable antibiotic 

To illustrate that the methodology described so far applies to a more challenging case and molecule, consider the example of a degraded antibiotic containing a substitute active pharmaceutical ingredient. LC–MS analysis showed that the sample was not a single compound (Figure 4). Obviously the first challenge was to focus on the correct chromatographic peak to be able to determine the active or substitute active pharmaceutical ingredient in the sample. The first hypothesis is that the main chromatographic peak comes from residual of active or is the main degradant. The main chromatographic peak is due to a compound with a nominal mass of 308, the mass-measured protonated molecule having 

Considering that the molecule might contain C, H, N, O, a maximum of three F and one S and setting a limit of 5 mDa for mass accuracy, the software offers 33 possible even-electron elemental compositions. The best match, in terms of mass accuracy, is C

F, with a DBE of –0.5. Chemically such a molecule is extremely unlikely, if not impossible. 

Analysis of the isotope cluster shows that the [M+H+1]

C contribution) represents about 18% of the [M+H]

C peak). The unknown compound therefore contains about 14–18 C atoms. Moreover, the [M+H+2]

  peak has an abundance of 7% relative to the 

S (with a relative ion abundance of about 4%) contributes to this peak. So the compound possibly contains one S atom. 

Wolff observes "You can more readily obtain information on the presence of an S atom, and even general information on the composition of the isotope cluster, using higher resolution instruments (60,000–100,000 FHHW) like FT or Orbitrap (Thermo Fisher)." Indeed, Figure 5 shows the MS spectrum from another compound, analyzed at 60,000 resolution, where you can clearly see 

  peak (shown in the figure), which is well-separated because of the simultaneous contribution from two 

 344.1227 is due to the contribution from one 

 344.1338 is due to the contribution from two 

C atoms in the molecule. The bottom spectrum is the simulated theoretical spectrum, which shows an excellent match for the isotope ratios and mass accuracy. 

"The isotope cluster analysis shows 14 to 18 C atoms and one S atom for the substitute, active pharmaceutical ingredient with 

 309. By setting the limits for the carbon atoms and forcing one S atom, the software calculation shows only three possible elemental compositions with a 5 mDa mass accuracy: C

S (4.5 mDa, 7.5 DBE). If I were less conservative and set the mass accuracy limit at 2 mDa, I would have realized only one hit. Generally, 2 mDa-accuracy is easily achieved on high-resolution mass spectrometers when the ion abundance falls within the detector's saturation limits." 

None of the three elemental compositions yielded a hit in the Merck Index, which lists all commercially available drug molecules. Consequently, the main peak in the chromatogram is most likely a degradant. Searching the chemical abstract, C

S (the elemental composition with the best accuracy) gave a match that corresponds to protonated benzylpenilloic acid (Figure 6), a known degradation product of penicillin antibiotics (like penicillin G).  

Figure 7 shows the LC–MS chromatograms for a degraded penicillin G standard and the counterfeit sample. The major peaks in the counterfeit sample are present in the degraded penicillin G standard. However, no residual penicillin G could be found in the counterfeit sample. The peaks at 1.9 and 3.4 min are well-known degradation products, both isomers of penicillin G: benzylpenillic acid (C

S). From this information you validly can infer that penicillin G (Figure 8) was the substitute active pharmaceutical ingredient in the counterfeit sample, before degradation. 

MS - The Practical Art

This month's column brings much of the previous discussion on how to interpret mass spectra to a practical examination of detecting substitutions in counterfeit pharmaceuticals.

Techniques for Structure Elucidation of Unknowns: Finding Substitute Active Pharmaceutical Ingredients in Counterfeit Medicines

For the seventh consecutive year, JEOL USA, Inc. (Peabody, Massachusetts), has received the Omega NorthFace Scoreboard Award in recognition of its commitment to providing exemplary service and exceeding customer expectations. 

"We at JEOL are proud of our continuous and measurable improvement over the years," said Patrick McGinley, Director of Service. "Supplying excellent service requires a corporate-wide commitment to satisfying the customer's needs." 

SGE and Australian Proteome Analysis Facility sign agreement

SGE Analytical Science (Austin, Texas) announced it has entered into a memorandum of understanding under an Australian government commercial-ready grant with Australian Proteome Analysis Facility, Ltd. (Sydney, Australia), a leading international proteomics service provider and technology developer for the development of applications of SGE's liquid chromatography and micro fabrication technologies. 

APAF and SGE plan to collaborate on the development and commercialization of new chromatography products for the protein biomarker discovery markets. APAF will publicise SGE's products under its services and skill-training programs to its customers and develop new ProteCol kits for a variety of applications. Once feasibility is established, customers will then purchase the kits directly from SGE.  

Spark Holland BV (Emmen, the Netherlands) received the ISO 13485:2003 certificate from its notifying body, the KEMA. This ISO quality system is designed for companies delivering to the clinical market. Spark Holland has registered its Symbiosis Pharma system as an FDA Class 1 Medical device and certified it as a CE IVD device. The Symbiosis system accepts raw biological samples, like plasma or urine, cleans them up, performs LC separation and then injects it directly into the MS.  

Waters and Northeastern University open laboratory

Waters Corporation (Milford, Massachuesetts) and Northeastern University today opened the doors of the Waters Mass Spectrometry Laboratory within the Barnett Institute of Chemical and Biological Analysis. The new laboratory is dedicated to studying protein shapes and characteristics to provide pharmaceutical and biotechnology innovators with the tools necessary to develop new treatment options for some of the world's deadiest diseases, like AIDS and cancer. 

Eksigent signs Korean distribution agreement

Eksigent (Dublin, California) announced a distribution agreement with Interface Engineering (Seoul, South Korea), a division of Bio-Medical Science Co., Ltd. The agreement covers sales and service of the NanoLC and ExpressLC HPLC product lines in Korea. 

Interface Engineering Co., Ltd. has been serving local clients and is a Korea's leading companies in the analytical field, especially in pharmaceutical and environmental research markets.

Departments

JEOL receives Omega Award; SGE and Australian Proteome Analysis Facility sign agreement; Spark Holland achieves ISO certification; Waters and Northeastern University open laboratory; Eksigent signs Korean distribution agreement

Peaks of Interest

							Although liquid chromatography (LC) is most commonly associated with analytical techniques that are found inside the laboratory, LC also has been used in industrial applications for separation of a wide range of products for decades. The technique recently has been applied to requirements in the pharmaceutical and biotechnology industries, both inside the laboratory and in industrial settings. With global sales of over $1.7 billion, the market for preparative LC has encountered a stellar growth over the past few years.  

Worldwide preparative and process chromatography demand.			

The steady growth of pharmaceutical and biotechnology research is continuing to drive strong demand for preparative chromatography, which has become a commonplace laboratory preparative technique for improving a wide range of analyses.  With the increase of the number of drug candidates reaching clinical trial phases, and full approval, non-analytical chromatography is seeing dramatic growth at the pilot and process scales. 

The preparative and process chromatography market, including prep HPLC and LPLC, flash, SFC, SMB, pilot, and process-scale chromatography, is likely to benefit from the FDA initiative to modernize the regulation guidelines of pharmaceutical manufacturing. Drug makers often have been reluctant to change manufacturing processes and equipment because of regulatory hurdles. Among other measures, an effort is being made to accommodate process changes that would support continuous process improvement and encouraging the use of modern tools. 

The foregoing data was extracted from SDi's upcoming Market Analysis and Perspectives report titled 

Purification Applications for Process and Preparative Liquid Chromatography.

 For more information, contact Glenn Cudiamat, VP of Research Services, Strategic Directions International, Inc., 6242 Westchester Parkway, Suite 100, Los Angeles, CA 90045, tel. (310) 641-4982, fax (310) 641-8851, e-mail 

lthough liquid chromatography (LC) is most commonly associated with analytical techniques that are found inside the laboratory, LC also has been used in industrial applications for separation of a wide range of products for decades. The technique recently has been applied to requirements in the pharmaceutical and biotechnology industries, both inside the laboratory and in industrial settings. With global sales of over $1.7 billion, the market for preparative LC has encountered a stellar growth over the past few years.

Preparative and Process Chromatography

Optimizing peak shape in capillary gas chromatography (GC) is essential for the consistent, accurate analysis of residual solvents (that is, trace levels of organic volatile chemicals present from manufacturing) in pharmaceutical compounds. Band broadening can result in peaks that can exhibit increased tailing and greater variability in peak area (1,2). This, in turn, can affect analysis accuracy by loss of resolution between compounds and failing data acceptance criteria (for example, replicate percent relative standard deviation [%RSD]). 

Various factors related to the instrument's set up have the potential to affect chromatography and peak area reproducibility. Slow detector responses can distort peak shapes (3,4). Peak shapes and peak maxima can be affected adversely by larger detector time constants, particularly for peaks with half-widths of 0.5 s or less. Also, low data sampling rates can reduce the reproducibility of the peak start, apex, and peak end, as well as the accuracy of a peak's shape (3). 

Stationary phase partitioning, longitudinal diffusion, and resistance to mass transfer in the mobile phase are the classic processes that affect peak broadening inside the column. The effects of these processes are expressed in the Golay equation, which can be employed to optimize performance based upon the type of column and carrier gas linear velocity (5,6). The Golay equation is defined as 

 is the longitudinal diffusion in the mobile phase, 

 is the resistance to mass transfer in the mobile and stationary phases, and 

 is the average carrier gas velocity. Stationary phase partitioning is of negligible contribution for columns with film thicknesses of 0.25 μm or less; however, above 0.5 μm, the partitioning effect can be significant enough to cause peak asymmetry (7). For any column, reversible solute adsorption to the stationary phase will be manifested as peak tailing (4). Also, analytes spread out to some extent as they diffuse through the mobile phase; however, all peaks should be affected equally by this gas-phase peak broadening (7,8).  

Proper column installation is also important to reduce problems with reproducibility and peak tailing. Excessive dead volume at the column fittings and leaking connections in the carrier gas lines, instrument bulkhead, and the column ends are contributors to band broadening and lack of reproducible peak areas (4,9,10). In addition, peak tailing can be particularly pronounced due to the vaporization of certain solvents in the injector port, especially when using a packed injector port, as opposed to a capillary injector port, for capillary column applications (2,10).  

If peak area reproducibility or peak tailing are still problematic after these factors have been eliminated or minimized, another factor to examine is the autosampler syringe. Unless all of the sample matrices being analyzed are very clean, nonvolatile residue and particulates eventually will build up within the barrel of the syringe, causing problems with injection reproducibility by degrading the fit of the plunger within the barrel (11). For this reason, most autosamplers are programmed to rinse the syringe with sample solvent between injections. However, more volatile solvents (for example, methanol and acetone) and certain sample matrices can increase the speed of residue accumulation even with rinsing between injections. The end result of slower or inconsistent injections is problematic reproducibility and issues with poor peak shape. Replacing or cleaning a syringe will solve the problem; however, replacing or cleaning a syringe several times during the course of sample analyses requires significant analyst time and effort. 

Method redevelopment might be required for implementing alternative sample solvents to reduce residue accumulation in the syringe plunger, or for changing the instrument configuration (for example, changing from packed to capillary injection port, changing the type of column used, and so forth). Given the time and cost associated with method redevelopment, an alternative means of improving syringe performance is desirable. 

In the case of the autosampler syringe, a simple way to address the issues of band broadening and reproducibility is to identify a material that can improve syringe lubrication, eliminating the need to replace syringes throughout a sample set. The ideal substance would be compatible with the analytical method in question; a liquid at room temperature; and sufficiently volatile to elute during the temperature program prescribed in the analytical method. For GC–FID residual solvents methods, one substance that is a viable candidate is 

-Methyl-2-pyrrolidone has been used in various environmental, petroleum, and pharmaceutical applications as a polar solvent for aromatic and C

+ aliphatic hydrocarbons, residual solvents, and parenteral pharmaceutical products (12–15). It is more viscous than water (1.65 cP) and has a dielectric constant of 32.2 at 25 °C (16). 

-Methyl-2-pyrrolidone is well suited for residual solvents analysis with GC–FID because it is miscible with water and organic solvents, it can solubilize various pharmaceuticals, and it has a boiling point high enough (202 °C) to be eluted after most common residual solvents (14,15,17). 

-methyl-2-pyrrolidone as a viable syringe lubricant to improve result reproducibility and peak shape, three GC–FID residual solvent methods that had exhibited repeated issues with poor standard %RSD and peak tailing were examined. 

Agilent 6850 and Agilent 6890 gas chromatographs (Santa Clara, California) with flame ionization detectors were used throughout these experiments. A packed or capillary injection port and an Agilent 10-μL autosampler syringe were used according to the individual analytical method. Syringe wash vials were set up for sample solvent in the wash 1 position and 

-methyl-2-pyrrolidone in the wash 2 position. Data were collected using Waters GlobalChrom Data System (GCDS) and Empower software (Milford, Massachusetts). The GC conditions and temperature program for each method are listed in Table I. 

Table I: GC conditions and temperature programs for the methods used in the study			

-Methyl-2-pyrrolidone (99.5% anhydrous) was purchased from Acros (Morris Plains, New Jersey). Purified water (Millipore, Billerica, Massachusetts, or equivalent), high performance liquid chromatography (HPLC)–GC grade acetone purchased from Burdick & Jackson (Muskegon, Michigan), and HPLC–GC grade methanol purchased from EMD (Gibbstown, New Jersey) were used as sample solvents. Acetone (99.9+% Chromasolv), methanol (99.8% anhydrous), 2-propanol (99.5% anhydrous), isoamyl acetate (98% reagent grade), 

-amyl acetate (99%), isoamyl alcohol (99+%), and dimethyl sulfoxide (99.9+% ACS reagent grade) were all purchased from Sigma-Aldrich (St. Louis, Missouri). 

-Amyl alcohol (99.2%) was also purchased from Acros. Reference standards of Lilly compounds pertaining to each method were obtained from Lilly Reference Standards Lab (Eli Lilly and Co., Indianapolis, Indiana). 

Columns and conditions used are listed in Table I. 

Samples were prepared in purified water at the following test concentrations: acetone, methanol, and 2-propanol each at 0.005 mg/mL; acetone, methanol, and 2-propanol each at 0.0005 mg/mL; and Lilly Compound A at 5 mg/mL. 

Samples were prepared in HPLC-grade acetone at the following test concentrations: isoamyl acetate, 

-amyl alcohol each at 0.25 mg/mL; isoamyl acetate, 

-amyl alcohol each at 0.00375 mg/mL; and Lilly Compound B at 35 mg/mL. 

Samples were prepared in HPLC-grade methanol at the following test concentrations: dimethyl sulfoxide at 0.025 mg/mL; and Lilly Compound C at 25 mg/mL.  

The three GC–FID residual solvents methods examined often exhibited poor peak tailing and standard peak area (SPA)%RSD during normal execution. In practice, each of the methods required the use of a new syringe every time a new sample set (for example, system suitability solutions, standards, samples) was analyzed because the syringe plunger regularly seized after a few injections. Analysts spent considerable time and effort to ensure the methods performed appropriately; additionally, poor peak tailing and SPA %RSD often required repeat testing. Peak tailing for these methods is defined as 

 is the time from peak start to peak maximum as measured at 10% peak height.  

The experiments for each analytical method using 

-methyl-2-pyrrolidone in a second syringe wash vial were compared with historical data that used only sample solvent as a syringe wash to assess method performance. Sample solvent blanks and quantitation limit samples were analyzed to obtain the elution profile for 

-methyl-2-pyrrolidone and examine any potential interference from this compound. In the case of Method 3, the dimethyl sulfoxide standard was used in lieu of a reporting limit sample because its response is near the limit of quantitation. Standard solutions were analyzed to obtain values for peak tailing and SPA %RSD. Method 2 does not use peak tailing for a system suitability criterion; therefore, only SPA %RSD values were evaluated. 

The peak shape for methanol and 2-propanol are critical for accurate quantitation due to the close elution of the two peaks. Injection of the sample solvent blank and quantitation limit sample showed no interference of 

-methyl-2-pyrrolidone with the peaks of interest (Figure 2). Peak shape for each analyte was improved (Figure 3), resulting in baseline separation of methanol and 2-propanol. Improved reproducibility for peak tailing and SPA %RSD were obtained when 

-methyl-2-pyrrolidone was used as a syringe wash (Table II). Of note is the marked improvement in methanol SPA %RSD, which failed the acceptance criterion frequently before the use of 

-methyl-2-pyrrolidone as a syringe wash. 

Table II: Method 1 peak tailing and standard %RSD results			

Injection of the sample solvent blank and quantitation limit sample showed no interference of 

-methyl-2-pyrrolidone with the peaks of interest (Figure 4). Improved results for SPA %RSD were obtained when 

-methyl-2-pyrrolidone was used as a syringe wash (Table III). Although historically, the method performed adequately in terms of reproducibility, SPA %RSD was improved approximately twofold by using 

Optimizing peak shape in capillary gas chromatography (GC) is essential for the consistent, accurate analysis of residual solvents in pharmaceutical compounds.

LCGC North America-06-01-2007

Current Trends in HPLC Column Usage

Techniques for Structure Elucidation of Unknowns: Finding Substitute Active Pharmaceutical Ingredients in Counterfeit Medicines

Peaks of Interest

Preparative and Process Chromatography

Improving Injection Consistency Using N-Methyl-2-Pyrrolidone as a GC Syringe Wash

The Perfect Method, Part I: What Is Your Goal?