Part II of this four-part series described the use of perfluorinated compounds as calibrants for electron ionization (EI) and chemical ionization (CI) mass spectrometry (MS), and described links between time/intensity files recorded by a data system, the calibration file, and the properly calibrated mass spectrum (1). Here, Part III describes calibrants that are used in higher masses accessed with other ionization methods. Many of the basic details of calibration remain the same, including the linkage between time/intensity files and the calibration file, and the fact that an external calibration (using a calibrant separately introduced into the ionization source of the mass spectrometer) is intrinsic to proper operation of the mass spectrometer.  

Kenneth L. Busch was among those curious about the origin of the chemical noise Ã¢ÂÂpeak at every massÃ¢ÂÂ in a FAB mass spectrum, musings that ultimately led to his development of charge-transfer derivatization in MS. Views expressed in this column are those of the author and not those of the National Science Foundation. Contact the author at: wyverners@yahoo.com.			

Electron ionization and chemical ionization are used for sample compounds from which a population of stable gas-phase molecules can be created through evaporation at (usually) elevated temperatures. Because sample volatility generally is inversely proportional to molecular mass (other factors also contribute), the mass range concordant with the use of EI and CI sources tops out at perhaps 1500 Da. With higher mass, and especially for most biomolecules, thermal stability is low, and direct evaporation of such compounds at higher temperatures cannot be achieved without decomposition. Accordingly, ionization methods other than EI and CI must be used to produce ions with higher masses extending to several thousand daltons. Details of the calibration process will change as the ionization method changes, as the breadth of the calibrated mass range increases, and as the type and use of the mass spectral data change. Figure 1 illustrates this latter point, plotting the fraction of the mass range from which analytical data typically is drawn against molecular mass. The situation is illustrated in the figure for a single stage of mass analysis; for tandem MS (MS-MS) analyses, the relationship differs. 

Figure 1. Generalized relationship between the fraction of the mass spectrum from which analytical information is drawn and the molecular mass of the compounds investigated.			

The perfluorinated calibrant FC-43 used for EI calibration contains in its mass spectrum an ion at 

 614, representing the high end of the useful mass range. As described in Part II, other perfluorinated compounds can be used to extend the calibration to still higher masses; these other calibrants have molecular masses higher than that of FC-43. Their use is predicated on the hope that the higher mass molecular ion and fragment ions could still be produced with discernible relative abundances. But as the molecular mass of the calibrant increases, the compound volatility decreases, and the relative abundances of the molecular ion and high mass fragment ions usually decreases. A strategy to reach higher calibrated mass ranges by increasing the molecular mass of the calibrant compound soon becomes inadequate.  

Using ionization methods such as liquid secondary ion mass spectrometry (LSIMS) and fast atom bombardment (FAB), and (in common usage today) electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI), the zenith of the mass range extends routinely to several thousand daltons (kDa), intermittently to several tens of thousand daltons, and exceptionally approaches the megadalton (MDa) mark. Calibration needs certainly change as analyses extend to these higher masses. Discussion here follows the chronological history of mass calibrants developed first for FAB and LSIMS, and then extends to calibrants developed (and still developing) for ESI and MALDI. Basic changes in strategies for instrument calibration itself (for example, no longer using an external calibration), the analytical subtleties of the higher mass territories, and missteps taken along the path to higher masses, are reserved for discussion in Part IV of this series.  

High mass calibrant compounds form higher mass ions through a process of clustering. Represented in a simple nomenclature, a calibrant compound of molecular mass M forms cluster ions M

 ranges from 1 to 50, and in which the charge can be either positive or negative. Ionic clusters consisting of cations (C) and anions (A), with a single ion excess of either the cation or the anion, also are used. Simply, and as reasonably expected, the relative abundances of cluster ions tend to decrease with higher mass, with some interesting exceptions for cluster ions of specific structures and exceptional stabilities. Mass intervals between ions of useful abundance in the mass spectrum of the calibrant compound are determined by the molecular mass of the calibrant in the case of a molecular cluster M

, or the mass of the CA unit in an ionic cluster. The desirability of using a calibration compound composed of monoisotopic elements remains, because as before, this characteristic maximizes the single-peak relative abundances of each of the calibrant ions. With the use of a clustering calibrant, however, rather than controlling the dissociative processes of a molecular ion, rational selection instead focuses on the tendency for the cluster ion to "stick together," based upon non-covalent interactions, or cemented with a singly charged charge carrier such as a proton, a halide ion, or a single excess cation or anion derived from the calibrant itself. Ideally, each molecular or ionic subunit should be robust and resistant to internal fragmentation, because these dissociative processes would detract from the relative abundances of the cluster ion. These desiderata are summarized in Figure 2. 

Figure 2. Generalized scheme for desirable characteristics of a clustering calibrant compound.			

The first applications of FAB/LSIMS in the 1980s, showcasing the newfound ability to ionize nonvolatile sample compounds with kilodalton masses, presented an immediate challenge to the standard procedures for instrument calibration. Assuming that the instrument response was stable, analysts extended the bar to masses much higher than the highest mass ion created from the calibrant compound. More importantly, external calibrants of the usual sort did not produce a mass spectrum with these new ionization methods. For a short time, the chemical noise that is a characteristic of the FAB mass spectrum (reflecting the sputter-induced chemistry of the liquid support matrix) was a blessing in disguise, as the masses of ions from the sample were established by manual count starting from ions of known masses. Such a mass spectrum was not mass calibrated externally through an instrument calibration then, but was internally calibrated. The origins of these near-baseline ions occupied the attentions of the chemically curious for some time, but their more immediate usefulness for mass counting is evident. Given an established consensus mass of any one ion in the mass spectrum and the resolved peaks at every integral mass, the integral masses of higher mass ions could be established by counting. Uncertainties in the mass assignments deduced in such fashion increased as the counting proceeded to higher masses, and as baseline peaks appeared with lower intensities. As we have described in previous columns, the isotopic envelope of higher mass ions begins to spread out over several integral masses. This is true of the background ions formed for a glycerol support matrix as it is for ions from sample molecules. Clearly, the practice of counting every background ion mass in a FAB or LSIMS mass spectrum to determine the masses of the sample-related peaks would not long suffice.  

What external calibrant compound might then be chosen for use in FAB and LSIMS? How might a calibrant be chosen so that calibrant ions appear with reasonable relative abundances at regular intervals within a wide mass range, and not be dependent upon evanescent background signals? The first practicable answer was serendipitously apparent in the liquid matrix used to dissolve and support the analyte molecules for FAB and LSIMS ionization. Glycerol (G), for example, forms a series of cluster ions in the positive ion mass spectrum G

 277, and so on, with decreasing relative abundances for higher mass cluster ions. For negative ions, analogous cluster ions (G

 are observed (see Figure 3). Glycerol cluster ions with 

 up to 15 and 20 could be observed at high detector gains, and the masses of these ions were, of course, predictable and precisely known. This clustering of the glycerol molecules, each cluster associated with a single proton, or deficient in a single proton, provided a means to calibrate the mass spectrum up to a mass of perhaps 2000 Da, with the higher intensity calibrant ions separated by the mass of glycerol itself (92 Da). Use of the glycerol clusters combined an internal and an external mass calibration process. A sample of pure glycerol could be used to create a separate external instrumental calibration file. The mass spectrum recorded from the sample itself would also contain ions corresponding to these glycerol clusters, and their appearance at the correct masses provides an internal check of consistency. 

Figure 3. Negatively charged cluster ions in the FAB mass spectrum of glycerol (G).			

Glycerol soon was augmented via exploration of the practical utility of many other liquid matrices; hundreds were explored eventually before common practice settled on a small number of generally suitable liquid matrices for sample preparation in FAB/LSIMS (2). Other support matrices also formed cluster ions of masses higher than the molecular mass that could be used for calibration. Soon, however, the limitations of using cluster ions from the matrix became evident, both as a practical matter in terms of the mass range but also the need to maintain an instrumental calibration separate from the matrix used for sample preparation, mass interferences that needed to be avoided, and the simple fact that an ideal support matrix would be itself transparent in the recorded mass spectrum. A change in the basic approach to calibration to embrace clustering calibrant compounds became apparent with the first use of the cluster ions generated from the liquid support matrix. Once this path was chosen, other potential calibrant compounds that produced cluster ions in their sputtered mass spectra were investigated, with a careful eye towards maximizing the intensity of the cluster ions, minimizing the width of the isotopic envelope for higher mass ions, and maintaining a separate external calibration process. Alkali halide salts (and mixtures of them) were established to provide cluster ions in both positive and negative ion FAB/LSIMS that could be used for calibration. The positive ion cluster ions C

 contained a single excess cation (C) that provided the single positive charge for the cluster; the negative ion cluster ions C

 contained a single excess anion (A). Cesium iodide is an especially useful calibrant compound. The integral mass of the cesium cation is 133 Da, and that of iodide anion is 127 Da, resulting in calibration ions that are spaced reasonably in both the positive and the negative ion mass spectra. Furthermore, each of these elements is monoisotopic, maximizing the intensity of the calibrant ion. Using cesium iodide as the calibrant, reliable mass calibration could be extended to 5 kDa and, with a little added effort, to 10 kDa. For more closely spaced calibrant ions, a mixture of CsI, RbI, and NaI also can be used, providing a wider variety of calibrant ions that contain mixtures of the three different cations. Cluster ions formed from cesium iodide have been observed to masses of up to 20 kDa, with an (almost) smoothly decreasing intensity curve (3–5). Deviations of observed ion intensities from the expected smooth abundance decay curve led to a brief flurry of research into "magic numbers" of ions in the gas phase; this topic is worthy of a separate future discussion. Calibration tables with the masses of calibrant ions from cesium iodide and other alkali halide mixtures are found in appendices of several mass spectrometry textbooks, and are available readily from manufacturers and from web resources. Such calibration tables also are preloaded into instrument software control systems.  

Once the strategy of exploring cluster ions for calibration to higher mass ranges became established, other potential calibrants were explored and described in the literature. Blaum et al. (6) described the use of atomic gold clusters for calibration; cluster ions of the form Au

 up to 50 provided a calibration to 10 kDa. Letzel et al. (7) describe an organic compound that provides stable cluster ions in both the negative and position ion modes of electrospray ionization, with the consequential suggestion that these ions could be used to provide mass calibration to a mass limit of 6 kDa. Konig and Fales (8) described organic salts of cesium that could be used for calibration in ESI up to a high mass limit of 10 kDa. Research into other calibrant compounds continues; the use of cesium iodide itself has the advantage of being simple and direct.  

As we foresee topics covered in the final Part IV of this series on calibration in MS, several issues become clear that complicate the "simple and direct" approach exemplified by cesium iodide. The first issue is a fact apparent in practice, and implicit in the simple relationship evident in Figure 1. Attempts to increase the breadth of the calibrated mass range are inconsistent with the fact that, in practice, the entire mass spectrum was not usually of interest. For example, a useable calibrated range of perhaps 2 kDa could be placed at many different places in the range, viz 1–3 kDa, 5–7 kDa, or 18–20 kDa. Seldom was a single mass spectrum from 0–20 kDa needed, so seldom is such a mass spectrum recorded or interpreted. For higher resolution mass measurements, even smaller relevant mass ranges were established. To coin a phrase, "concordant calibration" matches the calibrated mass range to the mass range from which the relevant analytical information is to be recorded. Selection of a concordant calibrant is part of intelligent experimental design. 

Secondly, in appreciation of basic analytical metrology, we note that calibration of mass spectrometers using either an external or internal calibration relies on linkage to the absolute mass scale based upon 

C can be created with any of several ionization methods, and such cluster ions can also be used for direct calibration of the mass spectrometer. We cover these and other topics in calibration in Part IV of this series. 

3. I. Katakuse, H. Nakabushi, T. Ichihara, T. Sakurai, T. Matsuo, and H. Matsuda, 

5. Y. J. Twu, C. W. S. Conover, Y. A. Yang, and E. A. Bloomfield, 

6. K. Blaum, G. Huber, H.-J. Kluge, and L. Schweikhard, 

7. M.C. Letzel, C. Agena, and J. Mattay, 

Articles

Columns

Part III of this four-part series continues the discussion of mass calibration.

Mass Spectrometry Forum - Mass Calibration: Cluster Calibrants for Higher Masses

Titration is a common analytical procedure used in modern laboratories. One of the first types of titration was invented by Joseph Louis Gay-Lussac, known as an author of "The Law of Combining Volumes." Today, titration is an analytical method that allows quantitative determination of a specific substance (analyte) in a sample by adding to it a second reactant solution of known concentration in carefully measured amounts until the reaction of definite and known proportion is completed. To be suitable for a determination, the end of the titration reaction must be observable. This means that the reaction should be monitored by an appropriate detection technique, for example, potentiometry, color indicators, pH, and so forth.  

One of the important applications of titration is analysis of molecular binding. In this case, an appropriate detection system provides quantitative information on the amount of titrant bound to its target and the nature of the binding. This allows the measurement of the binding isotherm, which represents the dependence of concentration of bound titrant on the total or free concentration of the titrant in solution. The binding isotherm then is used to calculate binding constants, stoichiometry, and the free energy of binding. Temperature dependence of the free energy is used to calculate the entropy and enthalpy of binding.  

The key element of titration analysis is the selection of an appropriate detector, which can provide quantitative information on the amount of titrant bound or reacted with the analyte. A range of techniques can be employed in titration analysis. The list of these techniques includes potentiometry, voltametry, electrical conductivity, isothermal titration calorimetery, UV/Vis absorbance, fluorescence, and others. However, none of these methods can serve as a universal detector for the binding of titrant to analyte. For example, fluorescence and UV/Vis absorbance require a change in optical activity of titrant or analyte in the binding as well as optical transparency of the solution. Therefore, titrations often require a complex sample preparation procedure such as extraction of analyte to make a solution with required optical transparency or other procedures such as attaching optical markers to titrant molecules, etc.  

High-resolution ultrasonic spectroscopy (HR-US) is an analytical technique based upon precision measurements of parameters (velocity and attenuation) of ultrasonic compression wave propagating through the analyzed sample. This technique allows direct probing of intermolecular forces and therefore can be used as a universal detector for titration analysis. Any change in molecular structure upon the binding affects intermolecular interactions in the sample and therefore can be detected with ultrasonic measurements. The measured ultrasonic titration profile can be recalculated into the binding isotherm. This technology is extremely sensitive, requires no markers, and can be used in non-transparent samples such as cell cultures or dispersions (for example, blood or milk). Another advantage of the HR-US titration technique is its ability to analyze molecules in their original state without immobilizing procedures or transferring into another environment. 

The key factor responsible for "ultrasonic visibility" of molecular processes is the resolution of the ultrasonic measuring devices, similar to a magnification power of telescopes in astronomy, which determines the visibility of stars. Novel principles of ultrasonic detection utilized in high-resolution ultrasonic spectrometers allow a tremendous increase in the resolution of ultrasonic instrumentation by several orders of magnitude when compared with the traditional ultrasonic techniques. Current applications of this technique include analysis of chemical reactions, conformational transitions in polymers and biopolymers, aggregation and gelation phenomena, particle sizing, phase transitions, stability of emulsions and suspensions, formation of micelles, ligand binding, composition analysis, and many others. 

Recently, capabilities of the HR-US technique have been expanded to titration analysis. HR-US titration spectrometers allow analysis of most chemical reactions and molecular bindings without or with minimum sample preparation. They do not require optical markers and optical transparency and use only a small volume of sample (down to 0.04 mL).  

 Figure 1. The HR-US Titration system: Titration module and HR-US 102 spectrometer are controlled by a PC using HR-US Titration Analysis software. 			

In this example, the Ultrasonic Titration system (Ultrasonic Scientific, Dublin, Ireland) (Figure 1) was used to analyze the binding of alamethicin to unilamellar DMPC (1,2-dimyristoyl-3-

-glycero-phosphocholine) vesicles. Alamethicin is a peptide antibiotic that forms transmembrane channels and regulates membrane permeability by acting as a monovalent cation ionophore. However, while the primary mechanism of antibacterial activity is not yet known, it is generally agreed that binding of peptide monomers to the surface of the target cells causes disruption, permeabilization, or disintegration of cytoplasmic membranes. It was proposed that depending upon the conditions, the peptides associate with membranes in two ways: they either adsorb parallel to the membrane surface (at low peptide/lipid ratios, P/L) or insert perpendicularly into the bilayer (at high peptide ratios), as illustrated in Figure 2. 

Figure 2. Ultrasonic titration profile of unilamellar vesicles of DMPC with pore forming peptide alamethicin in water. Change in ultrasonic velocity in solution caused by addition of the peptide is plotted as function of the molar ratio of alamethicin to DMPC (P/L). The curve for 17 Â°C corresponds to the binding with the gel membrane and for 33 Â°C, with the fluid membrane. (The gel to fluid transition temperature for DMPC membranes was about 23 Â°C.) 			

Binding of alamethicin depends upon the state of the lipid membrane, which could be in a fluid (at high temperature) or gel state (at low temperature). The transition temperature between the fluid and the gel states for the DMPC membranes is about 23 °C. In the current example, the binding was analyzed at two temperatures, 17 °C (gel state) and 33 °C (fluid state).  

The DMPC vesicles in water were prepared by extrusion and 1 mL of 7.54 mM DMPC suspension was loaded into the sample cell of the ultrasonic analyzer. The reference cell was filled with water. Solution of 5 mM alamethicin in ethanol was used as a titrant. The titrant solution was injected to the sample automatically in 1-μL steps. After each injection, the sample was mixed using double stirring systems that provided effective mixing within seconds. Ultrasonic velocity and attenuation in the suspension were monitored over the course of the titration. The same titration of DMPC vesicles was performed with just ethanol as titrant to determine the contribution of ethanol to ultrasonic parameters. The data were subtracted from the original titration curve and was corrected for dilution using Titration Analysis software (Ultrasonic Scientific). The resulting titration curves at two temperatures are given in Figure 2.  

The titration curve at 17 °C (gel state of the membrane) reveals two major stages of binding. The first stage is observed at low peptide to lipid (P/L) ratios, up to 0.0026 (or 1 peptide molecule per 380  lipid molecules). This stage shows a significant increase in ultrasonic velocity and therefore a decrease in compressibility of the system. This can be explained by adsorption of alamethicin molecules on the membrane surface. Molecular dynamic simulations (1) show that the helical structure of alamethicin is stabilized by the hydration water of the membrane. It is known that the strong binding of hydration water leads to a decrease in the compressibility. The adsorption of alamethicin molecules on the membrane surface also can reduce the movements of molecules of lipid, which will reduce compressibility of the membrane. 

Figure 3. Differential ultrasonic titration profile of unilamellar vesicles of DMPC with pore forming peptide alamethicin in water. The slope of the ultrasonic velocity curves (change in velocity per unit of P/L ratio) is plotted as function of the molar ratio of alamethicin to DMPC (P/L). The curve for 17 Â°C corresponds to the binding with the gel membrane and for 33 Â°C, with the fluid membrane. (The gel to fluid transition temperature for DMPC membranes occurs at 23 Â°C.)			

At high alamethicin concentration, P/L > 0.0085, the sound velocity shows a slow linear increase in the velocity with peptide concentration. This stage can be attributed to a solubilization (insertion) of alamethicin in the membranes. The change in the velocity upon the insertion is less in comparison with those for the adsorption when calculated per mole of peptide bound. This is seen clearly in Figure 3, which represents the slope of ultrasonic titration curve (change of ultrasonic velocity per unit of P/L ratio). The lower slope of velocity curve at the second stage can be attributed to a dehydration of peptide upon the insertion. In addition, the effect of peptide on compressibility of membrane also can be different compared with the first stage. Assuming that the initial (at P/L = 0/0.0015) and final (at P/L > 0.01) slopes of the ultrasonic velocity curve (Figure 2) correspond to the adsorption and insertion modes of the peptide binding, respectively, the percentage of alamethicin bound to the surface of the membrane and incorporated in the membrane was calculated and plotted in Figure 4. Figure 4 allows an estimation of the concentration corresponding to the beginning of incorporation of alamethicin into the membrane, the critical concentration for insertion or CCI, which is approximately 20 μM (at P/L = 0.0026) at 17 °C. 

Figure 4. Distribution of bound alamethicin molecules between the adsorbed onto the surface of DMPC membrane and the inserted into the membrane states at 17 Â°C as calculated from the slope of ultrasonic velocity. 			

At 33 °C (fluid membrane), ultrasonic titration curves (Figures 2 and 3) show a more complex profile of binding of alamethicin. The beginning of the binding is significantly different compared with the gel membrane, however, the last binding stage is the nearly the same for both fluid and gel membranes. A significant decrease in ultrasonic velocity in a narrow range of alamethicin concentration (2 m/s within the P/L range of 0–0.04) is an indication of a cooperative change in the lipid structure upon the peptide binding. One of possible reasons could be a fusion of lipid vesicles induced by the peptide. However, further studies of the binding process in a wider concentration range of lipids and at different temperatures could be essential to clarifying the mechanism of this process. These analytical tasks can be performed using the HR-US technique with expanded titration capabilities. 

HR-US titration analysis is a new, powerful tool in research and analytical laboratory work for quantitative measurements of different processes and compounds. This technique provides universal detection capabilities for molecular binding, as any binding affects intermolecular forces in the sample, and therefore, can be detected with ultrasonic measurements. Because the measurements do not require any optical transparency, molecular markers or other properties of solution and solutes, the complex sample preparation procedures in many cases become obsolete. 

1. P.D. Tieleman, H.J.C. Berendsen, and M.S.P. Sansom, 

 are with Arbeitsgruppe Komplexe Fluide, III Physikalisches Institut, Georg-August-Universitaet Goettingen (Goettingen, Germany).			

 are with Ultrasonic Scientific (Dublin, Ireland). E-Mail 

 is with the Department of Chemistry at University College Dublin (Dublin, Ireland).			

High-resolution ultrasonic spectroscopy titration analysis is a powerful new tool in research and analytical laboratory work for quantitative measurements of different processes and compounds. Here, the authors explore its potential.

New Capabilities of High-Resolution Ultrasonic Spectroscopy: Titration Analysis

Cleaning processes for pharmaceutical manufacturing equipment are inspected closely, because inadequate cleaning procedures can result in adulterated or contaminated products. Thus, the need to verify cleaning between manufacturing runs presents a special challenge. For cleaning verification, high performance liquid chromatography (HPLC) often is used to evaluate samples collected from manufacturing equipment. The cleaning verification process involves swabbing the surface and extracting the sample from the swab, followed by analysis using an HPLC system. Most laboratories use an HPLC system with an autosampler that allows sample analysis to take place unattended. Therefore, cleaning the pharmaceutical equipment, collecting and preparing samples for HPLC analysis, acquiring data, and processing can result in up to two days of lost production time. Other drawbacks to using HLPC methods for cleaning verification include incomplete analyte recovery from the surface and cross contamination resulting from handling and treatment of samples between swab collection and subsequent analytical work. Therefore, an ideal verification method for cleaning procedures would be a fast, automated, in-situ, multicomponent analysis of the entire surface. With such an approach, errors and inadequacies associated with surface sampling procedures can be eliminated and result in time and money savings. 

 Figure 1. Mid-IR spectra of compound 1 at different levels (bottom to top: 0.05, 0.17, 0.47, 0.78, 0.86, and 1.64 ÃÂ¼g/cm2) on metal coupons.

Recently, it was reported that in-situ spectroscopic techniques that employ middle-infrared (mid-IR) fiber optics and spectral imaging, for example, potentially could be used for the detection of small amounts of analytes from the surfaces of the equipment during the cleaning verification process (1â3). Thus, it is worthwhile to explore the feasibility of using mid-IR fiber optics for in-situ, noninvasive, accurate, and inexpensive cleaning verification. In the present study, the utility of such techniques in the submicrogram range is explored for the benefit of pharmaceutical manufacturing industries. 

Figure 2. Calibration curve for compound 1.

Grazing-angle spectra were obtained using a Remspec (Charlton, MA) SpotView mid-IR grazing-angle probe attached to a Bruker Optics (Billerica, MA) spectrometer with a Remspec external mercuryâcadmiumâtelluride (MCT) detector. The specially configured probe head illuminates a large spot on the sample surface (approximately 4.5 cm

) and maximizes the distance traveled through the surface layer by the IR beam before it returns to the detector. Spectra were collected across the range of 900â4000 wavenumbers (cm

), and a clean metal coupon (a flat stainless steel polished metal surface of area 4 Ã 4 in.) was used to collect a background spectrum. Sample spectra were recorded at 6 cm

 resolution using 100 scans with an approximate collection time of 0.5 min/spectrum). Opus 5.0 software from Bruker Optics was used for data acquisition. 

Figure 3. Comparison of predicted amount (using mid-IR) of compound 1 loaded on metal coupon. The actual amount loaded was determined by HPLC.

A calibration was built using the Quant 2 (Bruker Optics) package, an add-on to the Opus spectroscopy software. Quant 2 uses partial least square regression (PLS1). The calibration line generated was used to determine unknown amounts of the active pharmaceutical ingredient (API) loaded on the metal coupon. The samples also were prepared using a "smear" method. In the smear method, a known amount (0.1 mL) of a known solution of the target substance in methanol is dispensed onto a precleaned metal coupon. The solution then is "smeared" evenly across the surface and allowed to air dry. Alternatively, a solution of the target substance can be air-sprayed onto a pre-cleaned metal surface. 

Figure 4. Mid-IR spectra of compound A at different levels (bottom to top: 0.07, 0.22, 0.32, 0.54, 0.79, and 1.12 ÃÂ¼g/cm2) on metal coupons.

The coupons were dried, and sets of probe spectra were collected from at least six different locations on the metal coupon. The coupons were thoroughly washed, the washings analyzed by HPLC, and the "true" total surface concentration calculated. Reversed-phase HPLC methods developed in-house were used to determine the amount of APIs recovered using external standard. In the present study, three neat APIs (compounds 1, A, and AE) as well as a mixture of API (compound A) and excipients (Avicel, starch, lactose, magnesium stearate, and cabosil) were analyzed. A pure API stock solution in HPLC-grade methanol was prepared and then diluted to the microgram-per- milliliter range. The range of concentrations of these analytes used in the calibration was 0.05â1.5 Î¼g/cm

. For each compound evaluated, a calibration equation was generated and the concentration of few unknown samples was determined using the calibration to validate the calibration process. The amount of API determined using the spectrometric calibration was compared with that obtained from HPLC analysis. 

Figure 5. Calibration curve for compound A as neat analyte.

In the Fourier transform-infrared experiment, the signal amount acquired from a sample depends upon both the area illuminated by the IR beam and the path length traveled by the mid-IR radiation through the sample. The absorptivity of the samples for the concentration range used appears to be insignificant as the sample was deposited as a thin layer on metal coupon, as indicated by linear calibration plots. The grazing-angle head uses carefully aligned mirrors to deliver the mid-IR beam to the sample surface at the grazing angle (approximately 80Â° from normal) (4) to collect the reflected beam, and to return it to a mid-IR detector (in this case, an MCT detector). The signal is delivered from the spectrometer to the head and returned to the MCT detector by IR-transmitting fiber optic cables (measuring approximately 2-m long). The specially configured head illuminates a large spot on the sample surface (about 4.5 cm

) and maximizes the distance traveled through the surface layer by the IR beam before it returns to the detector. That combination of factors increases the sensitivity and signal-to-noise performance of the probe by several fold when compared with conventional reflectance methods using a mid-IR beam normal to the sample surface. 

Figure 6. Comparison of predicted amount (using mid-IR) of compound A loaded on metal coupon. The actual amount loaded was determined by HPLC analysis.

It was found that the measurements made on the samples prepared following the smear technique were not consistent. This probably could be due to inhomogenity of samples on the metal coupon. Therefore, only limited measurements were made on the samples prepared using the smear technique. However, consistent spectra were obtained on the samples prepared using spray technique. 

The recovery of compound l from metal coupon is nearly quantitative when the amount is greater than 0.4 Î¼g/cm

Figure 7. Calibration curve for compound A in the presence of excipients.

Compound A as neat API as well as in the presence of excipients from metal coupon can be determined quantitatively when the concentration is above 0.3 Î¼g/cm

Figure 8. Comparison of predicted amount (using mid-IR) of compound A in the presence of excipients loaded on metal coupon. The actual amount loaded was determined by HPLC analysis.

For compound AE, a calibration curve was generated in the concentration range 0.3â1.12 Î¼g/cm

. An unknown amount of compound AE was loaded on metal coupon and the mid-IR spectra were measured. The predicted values (spectra were collected for each sample on the metal coupon from eight locations and averaged) using the calibration curve were found to be in good agreement with those obtained from HPLC analysis (true values) as shown in Table I. 

Table I. Actual versus predicted values for compound AE

A mid-IR fiber optic probe with a grazing-angle head can reliably measure small amounts of organic contaminants on metal surfaces with loadings below 1 Î¼g/cm

. APIs can be analyzed in the presence of excipients on the same surface. There is the potential to build comprehensive calibrations across different surfaces. Thus, a "universal" calibration for a given compound on a range of surfaces might be possible. The technique is rapid, easy to use, and provides a direct, in-situ measurement. The probe requires adjustment, however, to accommodate curved surface geometries of manufacturing equipment. A more reliable sample application technique to generate calibration equations is needed as well. 

The authors wish to thank Raju Vegesna for helpful discussions and encouragement, Bruker Optics for providing instrumentation, and Dana Kelley at Bruker Optics and Mary Thompson at Remspec for technical help. 

1. N.K. Mehta, J. Goenaga-Polo, S. Hernandez-Rivera, D. Hernandez, M. Thompson, and P. Melling, 

3. K. Payne, W. Fawber, J. Faria. J. Buaron, R. DeBono, and A. Mahmood, 

The Role of Spectroscopy in Process Analytical Technologies

 (John Wiley & Sons, Hoboken, NJ, 1986), p. 194.

 is a senior scientist, at Novartis Pharmaceuticals Corporation, Analytical Research & Development (East Hanover, NJ). E-mail: 

The authors describe an in-situ cleaning verification technique that promises to be faster and more efficient than traditional methods, which use swab sampling followed by HPLC analysis. The technique is based upon FT-IR spectroscopy in the middle-infrared (mid-IR) range using reflection at a grazing angle.

Quantification of Active Pharmaceutical Ingredients on Metal Surfaces Using a Mid-IR Grazing-Angle Fiber Optics Probe — An In-Situ Cleaning Verification Process

Spectroscopy-10-01-2005

Mass Spectrometry Forum - Mass Calibration: Cluster Calibrants for Higher Masses

New Capabilities of High-Resolution Ultrasonic Spectroscopy: Titration Analysis

Quantification of Active Pharmaceutical Ingredients on Metal Surfaces Using a Mid-IR Grazing-Angle Fiber Optics Probe — An In-Situ Cleaning Verification Process