Analytical Applications

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Pharmaceutical Technology, Pharmaceutical Technology-11-01-2011, Volume 2011 Supplement, Issue 6

A Technical Forum Moderated by Patricia Van Arnum, featuring contributions from PerkinElmer, BioTools, Chiral Technologies, Shimadzu Scientific Instruments, GE Analytical Instruments, and Waters.

Developing methods to analyze drug substances and finished drug products is crucial for ensuring the quality of pharmceutical products. Several industry experts discuss applications in pharmaceutical analysis. Jerry Sellors, IR business manager at PerkinElmer, examines attenuated total internal reflectance sampling in Fourier tranform–infrared spectroscopy. Bo Wang, research scientist, and Laurence A. Nafie, chief technology officer, both with BioTools, and Elena Eksteen, senior business manager, business development and planning at Chiral Technologies, discuss vibrational circular dichroism technology in determining the absolute configuration of enantiomers. Robert H. Clifford, PhD, industrial business unit manager at Shimadzu Scientific Instruments, discusses ultraviolet–visible spectrophotometry in determining the quantitation limit of residual samples in cleaning validation. Richard Godec, new product development manager, Jon Yourkin, pharmaceutical market manager, and Kevin Aumiller, product manager, at GE Analytical Instruments, examine on-line total organic carbon analysis for pharmaceutical water. St. John Skilton, PhD, senior marketing manager, business operations, LSD, pharmaceutical life sciences at Waters, explains the role of hydrogen/deterium exchange with mass spectrometry in biopharmaceutical analysis.

Attenuated internal reflectance sampling in FT–IR

Jerry Sellors, IR business manager at PerkinElmer

Most pharmaceutical laboratories use a Fourier transform–infrared (FT–IR) spectrometer for testing materials. Mid-IR spectroscopy provides rapid and highly specific pharmaceutical ingredient identity testing. IR spectroscopy is used for quantitative and qualitative analysis of solids and liquids, but the most common use for pharmaceuticals analysis is identity confirmation of powdered ingredients. Three common sampling techniques are used: two involving transmission sampling and a third involving attenuated total internal reflectance (ATR).

One approach in transmission sampling is the alkali halide disk method (often referred to as the KBr method), which involves dispersing the sample in a nonabsorbing matrix (e.g., potassium bromide [KBr]) and pressing the mixture into a semitransparent disk to be measured in transmission by passing the IR beam through the disc. Alternatively, in another transmission method, the sample is mixed with a mineral oil and ground into a paste that is pressed into a thin layer between two IR-transparent windows and measured. Both techniques require manual sample preparation, are time-consuming, and may be prone to error. Among all the pharmacopeias, however, these two methods for IR solids sampling are most commonly used. Despite advances in FT–IR technology to provide increased sensitivity, reproducibility, and reliability, developments in sampling apparatus for these techniques have been unremarkable.


Advances in transmission sampling have been slow to develop in part because of the use of another approach, attenuated total internal reflectance (ATR) techniques for IR measurement of solids. With ATR, the sample is pressed into contact with a high refractive index crystal, such as germanium or diamond, and the sample is measured by reflection (see Figure 1). ATR systems often are designed as a central component of the overall FT–IR system. Under ATR, users can more easily acquire good quality IR data from a wide range of samples with minimal sample preparation. ATR technology has improved with respect to crystal design and hardware coupling with FT–IR systems as well as with respect to the software performing automated system performance, suitability, and contamination checks. ATR crystals also can be coupled with mid-IR fiber optics and light-guide systems, where improved sample accessibility is required.

Figure 1 (FT–IR): Schematic for attenuated total internal reflectance for infrared (IR) measurement of solids. (FIGURES 1–3(FT–IR) ARE COURTESY OF THE AUTHOR)

The level of acceptance of the ATR technique among the different pharmacopeias is somewhat mixed. For example, the US Pharmacopiea recognizes the technique as an acceptable alternative, but many other pharmacopeias do not mention ATR. The responsibility is with the user to demonstrate equivalence when this method is chosen to replace existing transmission methods.

The acceptance of ATR in regulated environments requires a general understanding of ATR because new sources of variation not encountered with conventional transmission measurements can affect the reliability of results. ATR is a reflection technique and can show distinct surface and optical geometry effects that may need to be characterized (1). For example with ATR, IR spectral-band intensities generally change with increasing force applied, and the effect is confounded by an increase in sample penetration depth across the wavelength range measured. This variability can affect quantitative and qualitative IR measurements, which rely on relative band intensities. An example of this can be seen in the ATR spectra of kaolin (see Figure 2) recorded at two different contact pressures, where a silicon–oxygen band in the spectrum of the mineral has a significant shift attributed to deformation of the crystal lattice. Assessing the effects of such variability is important for some materials, where altering pressure can change the degree of sample crystallinity and polymorphic form. Consequently, some ATR devices are designed to provide real-time display of force and spectral intensities before recording the IR spectra so that both can be checked before measurement. Pharmaceutical packaging materials also are frequently analyzed using ATR.

Figure 2. (FT-IR): Attenuated total internal reflectance spectra of kaolin at different sample pressures. A is absorbance.

In cases where oriented samples are presented to the system, care should be taken to understand the effect or orientation on the FT–IR spectra used in the analysis. This orientation effect is particularly true with molded or extruded polymers. Figure 3 shows the spectra of the surface of a polylactic acid where the only difference between the two measurements is a rotation of the sample on the ATR device. This difference is large enough to cause problems with routine measurements.

Figure 3: Attenuated total internal reflectance spectra of polylactic acid with rotation of sample. A is absorbance.

As IR use increases for routine measurements of pharmaceuticals, the acceptance of ATR among the pharmacopeias is likely to increase. Using ATR as an alternative to existing transmission methods requires showing evidence of suitability. Controlling and understanding the sources of measurement variation are important. IR instrument packages are being expanded with more sophisticated software routines to improve confidence in ATR results and knowledge-based offerings to assist the user in method development and validation.

FT–IR reference

1. R. Spragg, "Contact and Orientation Effects in FT–IR ATR Spectra, Spectrosc., (Aug. 1, 2011).

Determination of the absolute configuration of resolved enantiomers

Bo Wang is a research scientist, BioTools, and Laurence A. Nafie is chief technology officer, BioTools and distinguished professor emeritus, Syracuse University. Elena Eksteen is senior manager, business development and planning, Chiral Technologies

During the small-molecule drug development process, chromatographic resolution is an effective way for separating racemic compounds into single enantiomers for use in bioassays and toxicology studies. Chromatography also is a tool that can be used to establish pharmacokinetic and toxicological properties of candidate drug compounds (1). Vibrational circular dichroism (VCD) technology is an important tool for determining the absolute configuration (AC) of enantiomers and is used by pharmaceutical companies for molecular structure characterization of chiral molecules. Knowledge of the AC is vital in drug discovery, development, and the regulatory requirements for investigational new drug submissions (2).

VCD is the small difference in the IR absorbance of a chiral molecule for left versus right circularly polarized light. The determination of AC using VCD is a method for assigning the absolute stereochemistry of chiral molecules. It supplements, or in some cases, replaces the previous gold-standard method of anomalous X-ray diffraction that requires a pure single crystal of one enantiomer of the chiral molecule. VCD requires no crystallization and no chemical modification or derivatization of the chiral molecule. Assignments of AC are made by comparing the solution-state VCD and IR absorbance spectra to the corresponding quantum chemical calculations of the same spectra. When the VCD spectra agree, the AC chosen for the calculations is the same as that of the sample. If the signs of the calculated VCD are opposite to that of the measured VCD, the sample has the opposite AC. In addition, valuable information about the conformation or conformations of the chiral molecule in solution also may be obtained. Instrumentation for the measurement of VCD (Chiral IR-2X, BioTools) and software (Gaussian 09) for the calculation of VCD are commercially available as are services for performing AC determinations using VCD.

Figure 1 (Chiral): Separation of a racemic mixture. (FIGURES 1–3 (CHIRAL) ARE COURTESY OF THE AUTHORS)

Case study. A client requested enantiomer separation of a racemic mixture to obtain pure enantiomers needed for toxicology studies. In preparation for clinical trials, the client also requested AC measurements for an IND submission. The racemic compound was screened against several chiral stationary phases to identify a method appropriate for the separation of 50 to 100 g of the mixture. Figure 1 (Chiral) shows the separation of the racemic mixture using a Chiralpak IA column (Chiral Technologies).


Figure 2 (Chiral): IR absorbance (bottom), vibrational circular dichroism VCD (middle) and VCD noise spectra (top) of the two enantiomers before baseline subtraction. The two enantiomers show identical IR spectra, as expected, but opposite-signed VCD spectra for every IR band.

Using the conditions of the separation method for scale-up, 80 g were separated, and pure enantiomers were isolated for toxicology studies and for VCD-based AC measurements of the pure enantiomers. VCD spectra of the resolved enantiomers were measured using a Chiral IR-2x instrument (BioTool) The IR and VCD spectra are shown in Figure 2 (Chiral). Figure 3 (Chiral) demonstrates the comparison of experimental to theoretical spectra.

Figure 3 (Chiral): Comparison between observed vibrational circular dichroism (VCD) for one enantiomer and the corresponding calculated VCD for the S,S-configuration of this enantiomer. The agreement in signs between the two spectra yields an absolute configuration assignment of S,S for the enantiomer.

Chiral references

1. K. Valko, Eur. Pharm. Rev. Issue 5, 40 (2010).

2. H. Yanan et al., Appl. Spectrosc. 65 (7), 699–723 (2011).

Cleaning validation using UV–VIS spectrophotometry

Robert H. Clifford, PhD, industrial business unit manager, Shimadzu Scientific Instruments

Maintaining quality control and product safety is key to pharmaceutical manufacturing. Cleaning manufacturing equipment is essential for preventing contamination and cross-contamination. Contaminants from the environment must not get mixed with product ingredients, and residual substances adhering to manufacturing equipment cannot contaminate the next product in line for processing.

To verify these requirements, the cleaning process must be validated. The quantitation limit of an analytical instrument is the value at which residual samples can be quantitated. To determine whether the instrument to be used for cleaning validation has the resolution or sensitivity to detect to the permissible level of residual substance, it is important to determine the quantitation limit. In this example, the quantitation limit was determined using ultraviolet–visible (UV–VIS) spectrophotometry using samples consisting of one sample of an undisclosed detergent, Detergent A, often used for cleaning in the pharmaceutical field, and samples of acetylsalicylic acid and isopropylantipyrine.

One method of obtaining the quantitation limit is to determine the concentration value that corresponds to the absorbance (10 times the noise level). This method involves measuring the absorption spectrum of a standard sample and noting the wavelength of the greatest absorption peak. The next step is to measure the absorbance values at the wavelength of the greatest absorption peak using several samples of known concentration. The slope of the calibration curve is determined from the relationship between the concentrations of the samples and the respective absorbance values. Finally, repeat measurement of a blank sample (i.e., dilute solvent) is conducted to obtain the standard deviation. The quantitation limit is calculated from the slope of the calibration curve and the value equivalent to 10 times the standard deviation. Determination of the quantitation limits of Detergent A, acetylsalicylic acid, and isopropylantipyrine according to this method are presented in Figures 1–4 (UV-VIS).

Figure 1 (UV–VIS): Absorption spectra of detergent A with sample concentrations of 100 mg/L and 10 mg/L. (FIGURES 1 (UV-VIS) IS COURTESY OF THE AUTHOR)

Figure 1 (UV–VIS) shows the absorption spectra of detergent A with sample concentrations of 100 mg/L and 10 mg/L. Figure 2 (UV–VIS) indicates the calibration curve at a measurement wavelength of 225 nm. The quantitation limit for detergent A is determined to be approximately 0.16 mg/L.

Figure 2 (UV–VIS): Calibration curve at a measurement wavelength of 225 nm.

Figure 3 (UV-VIS)shows the absorption spectrum of acetylsalicylic acid methanol solution. The sample concentrations from higher to lower absorbance values are 400, 160, 80, 40, 20, and 8 mg/L. After 10 repeat measurements of a blank sample, the quantitation limit for acetylsalicylic acid is determined to be 0.42 mg/L.

Figure 3 (UV–VIS): Absorption spectrum of acetylsalicylic acid methanol solution.

In Figure 4 (UV-VIS), the absorption spectrum of isopropylantipyrine methanol solution is shown. The sample concentrations from higher to lower absorbance values are 80, 32, 16, 8, 4, and 1.6 mg/L. After 10 repeat measurements, the quantitation limit for isopropylantipyrine is determined to be 0.092 mg/L.

Figure 4 (UV–VIS): Absorption spectrum of isopropylantipyrine methanol solution.

In conclusion, the measurement results for detergent A, acetylsalicylic acid, and isopropylantipyrine illustrate the method of calculating quantitation limits based on measurement conducted using a UV-VIS spectrophotometer. Obtaining the quantitation limit makes it possible to verify the lower limit of residual substances and detergent that can be quantitated. UV-VIS spectrophotometry is a justifiable addition to cleaning validation tools alongside total organic carbon analyzers and high-performance liquid chromatographs.

Real-time TOC analysis

Richard Godec is new product development manager, Jon Yourkin is pharmaceutical market manager, and Kevin Aumiller is product manager, all with GE Analytical Instruments

In January 2011, FDA released Guidance for Industry: Process Validation: General Principles and Practices, which outlines the current regulatory thinking for process validation and control (1). It fully embodies the application of quality by design (QbD) and process analytical technology (PAT) methodologies that pharmaceutical companies can use to validate manufacturing processes. The new guidance also highlights the increased relevance of pharmaceutical manufacturing standards developed with voluntary consensus organizations, such as ASTM International. Of the 15 references within the guidance document, five refer to ASTM standards detailing best practices for process validation activities.

To understand why FDA is promoting ASTM standards in pharmaceutical manufacturing, one need only turn to federal law. The National Technology Transfer & Advancement Act requires governmental bodies to, wherever possible, adopt volunteer consensus-based standards to carry out policy. ASTM in collaboration with pharmaceutical companies, equipment vendors, process-design professionals, professional societies, and FDA, created the ASTM Committee E55 on Manufacture of Pharmaceutical Products and dedicated it to the development of new pharmaceutical manufacturing consensus standards in the spirit of this law. It is within this context that a number of high-level guidance and lower-level technology practice standards were developed and released (2).

In October 2010, the ASTM E55 committee promulgated a new standard practice, E2656, "Real-time Release Testing of Pharmaceutical Water for the Total Organic Carbon Attribute" (3). This real-time testing process is an example of QbD or PAT process that can be implemented to improve quality and reduce costs associated with traditional laboratory sampling. The new E2656 standard provides detailed suggestions and recommendations to use on-line total organic carbon (TOC) analysis for releasing water to the TOC attribute into the manufacturing processes. To achieve improved product quality, minimize risk, and to reduce manufacturing costs, many pharmaceutical companies are moving laboratory testing of TOC to real-time testing on line. This transition has important aspects, including a greater process understanding, strict control of key process variables, and the establishment of a process-capability specification to ensure the finished products meet quality requirements and regulatory guidelines.

The need for the standard was established based on prior problems encountered by pharmaceutical companies that had implemented real-time water release based on TOC. Previous problems included false positives from inappropriate TOC instrument selection, poor project implementation, insufficient understanding of the technologies, and insufficient knowledge to ensure the proposed approach would meet regulatory expectations. Over 30 subject matter experts developed the standard to provide the critical tools and solutions to these issues within a structured implementation process.

ASTM E2656 allows users to align with pharmacopoeia requirements for TOC analysis while also meeting the global regulatory compliance expectations of greater process understanding and control that are called for in relevant federal regulations (1, 4, 5). The standard is divided into six sections to assist in implementation as outlined:

  • Technical evaluation: determine the appropriate TOC technology based on thewater-system characteristics and intended use of the device.

  • Risk assessment: determine the appropriate placement of on-line TOC instruments using risk-assessment tools.

  • Data quality: qualify all on-line TOC instruments, compare the on-line method to legacy laboratory methods, and validate the relationship between the installation location and the relevant points of use.

  • Implementation strategies: develop processes to incorporate on-line TOC devices into quality, manufacturing, and maintenance systems.

  • Continuous-verification procedures: use statistical tools and ongoing verification strategies to assess process control.

  • Continuous-process improvement: Use the process knowledge gained from monitoring to make incremental improvements to the system.

Within each section of E2656, practical tools and best practices are identified to assist in the successful transition to on-line release testing. Some key elements of the process are outlined in Table I.

Table I: Tools for on-line real-time release testing of pharmaceutical water using total organic carbon (TOC) analysis based on ASTM E2656 (2).

In conclusion, the ASTM E2656 standard provides a structured process to efficiently implement on-line TOC analyzers for process control and real-time release testing. The standard aligns with regulatory guidance and, as a consensus-based practice, is supported by pharmaceutical manufacturers and other key players. ASTM E2656, provides a practical framework for improved water-system control.

TOC references

1. FDA, Guidance for Industry: Process Validation: General Principles & Practices (Rockville, MD, Jan. 2011).

2. Public Law 104–113, "National Technology and Transfer Advancement Act of 1995" (Washington, DC, 1995).

3. ASTM International, ASTM Standard E2656, "Standard Practice for Real-time Release Testing of Pharmaceutical Water for the Total Organic Carbon Attribute" (West Conshoken, PA, 2010), DOI:10.1520/E2656-10.

4. USP 34NF 29 General Chapter <643>, "Total Organic Carbon," p. 251.

5. Eur.Ph (6th ed.), 2.2.44, "Total Organic Carbon in Water for Pharmaceutical Use," 01/2008:20244, p. 71.

Hydrogen/deuterium exchange with MS

St. John Skilton, PhD is senior manager, business operations, LSD, pharmaceutical life sciences, Waters Corp.

Invention, adaptation, and innovation have played a role in developing commercial hydrogen/deterium (H/D) exchange (HDX) systems. HDX as a technique was pioneered by Lindstrom Lang in the 1940s. The basic principle is straightforward: place a molecule in a solution of D2O and measure how many–and how quickly–hydrogen atoms exchange with deuterium atoms. From this measure, the higher order structure of the molecule, typically a protein, could be inferred. Some regions of a protein would have faster uptake than others, and regions can be compared to provide a detailed picture of the three-dimensional structure of the protein. This allows proteins in different states or with mutations to be compared and correlated to biological activity. Lang used crude techniques, such as scintillation meters, to time the different rates at which deuterated versus nondeuterated samples fell through a liquid-filled tube under the action of gravity. The biotechnology industry has since adopted this method as a routine technique (1, 2).

Although HDX had previously been performed with high-performance liquid chromatography (HPLC), HPLC had weaker separation power. For HDX with mass spectrometry (MS), ultra-performance liquid chromatography (UPLC) separations allow more detailed measurements, an important consideration for use with proteins as large as antibodies. UPLC, therefore, improved the efficiency of HDX by having a higher degree of separation concomitant with robustness One challenge in quantifying deuterium uptake was the dynamic nature of the process. Deuteration is not a one-way street: molecules in solution will exchange back and forth dynamically, so in order to measure the process accurately, the exchange had to be chemically quenched to a pH of 2.5 (for proteins) and the analytical separation simultaneously cooled to 0 °C to manage the "back" exchange. Operating at such cold temperatures was not the sort of treatment chromatographic systems were originally designed for, so innovation was needed was to make a refrigerator unit integrated with the UPLC system. Having a cold pathway manages the reversal of the deuteration and brings the chromatographic separation as close to the detector as possible (3).

Figure 1 (HDX/MS): A depiction of the relative deuterium uptake for interferon helps one visualize and interpret the higher order protein structure related to conformational change. The uptake measurements are made at the peptide level for multiple time points across the experiment. Each uptake measurement is superimposed on the 3D structure of the protein, typically obtained from an X-ray representation. (FIGURE 1 (HDX/MS) IS COURTESY OF THE AUTHOR)

Another element in advancing HDX with UPLC/MS was the availability of a detection system that could cope with the complexity of the analysis and be able to make reproducible, quantitative measurements. The detection technique most appropriate for this need was an existing methodology applied in a new way: mass spectrometry with MSE (Waters Corp.). In MSE methodology, peptides are detected intact and then fragmented in rapid succession in the collision cell of the mass spectrometer, dozens of time per second (1). This method is possible in QTof and SYNAPT spectrometers, which are hybrid, tandem mass spectrometers (Waters Corp.) Therefore, for the same (peptide) ion, an accurate mass and an amino acid sequence is available. From the differences in masses between deuterated and undeuterated peptides, a precise determination of uptake, as well as proof of assignment, can simultaneously be made. This gain in information content allows the precise location of conformational changes (4, 5, 1).

As more complex proteins are analysed, it also becomes increasingly useful to include ion-mobility separations to disentangle the data (6, 2). Ion mobility separations are built in to SYNAPT mass spectrometers and have helped to further advance the HDX/MS applications area (7).

In HDX studies, data are produced across multiple time points, multiple species, and with replicates. Curating this data manually is not time-efficient and requires expert interpretation. The interpretation is a repetitive process that requires counting and measuring spectra, so the process can be automated with some in-built intelligence. Software (DynamX, Waters Corp.) is designed to systematically select spectra with predetermined criteria and measure the mass change of the deuterated form. The software automation was greatly simplified by having sharper peaks and better separation with UPLC, and the comprehensive nature of the MSE detection. This automation, along with the capability to sort and display data, has been an important advance.

HDX/MS References

1. I.A. Kaltashov et al., J. Am. Soc. Mass. Spectrom. 21 (3), 323–337 (2010).

2. R.E. Iacob, J.P. Murphy, and J.R. Engen, Rapid Commun. Mass Spectrom. 22 (18), 2898–2904 (2008).

3. T.E. Wales, Anal. Chem. 80 (17), 6815–6820 (2008).

4. J. Engen, Anal. Chem. 81 (19), 7870–7875 (2009).

5. Z. Jianming et al., Nature 463 (7280), 501–506 (2010)

6. K.D. Rand et al., "Gas-Phase Hydrogen/Deuterium Exchange in a Traveling Wave Ion Guide for the Examination of Protein Confirmations, Anal. Chem. online, DOI 10.1021ac901897x, Nov. 18, 2009.

7. R.E. Iacob et al., Proc. Natl. Acad. Sci. USA 106 (5), 1386–1391 (2009).