Raman spectroscopy method validation

In ocular applications, Raman spectroscopy can quickly and objectively assess composite lutein and zeaxanthin concentrations of macular pigment using spatially averaged, integral measurements or images that quantify and map the complete MP distribution with high spatial resolution. Importantly, both variants can be validated with HPLC methods in excised human eyecups and in animal models. 

When investigating opaque or transparent samples, where the laser light can penetrate the surface and be scattered into deeper regions, Raman light from these deeper zones also contributes to the collected signal and is of particular relevance with non-homogeneous samples, e.g., multilayer systems or blends. The above equation is only valid, if the beam is focused on the sample surface. Different considerations apply to confocal Raman spectroscopy, which is a very useful technique to probe (depth profile) samples below their surface. This nondestructive method is appropriate for studies on thin layers, inclusions and impurities buried within a matrix, and will be discussed below. 

Besides the methods given in Table 6-1 and Table 6-2, several others are available to determine dinitrophenols in biological and environmental samples. Immunoassay methods with sensitivities comparable to those of the conventional methods given in Tables 6-1 and 6-2 are noteworthy (Bush and Rechnitz 1987 Huang et al. 1992 Kusterbeck et al. 1990 Wannlund and DeLuca 1982). However, methods based on antibody sorption have not yet been validated on samples derived from environmental and biological sources, so it is not clear what methods of clean-up are necessary prior to quantitation of dinitrophenols. Some of the other detection methods with superior sensitivities that can determine dinitrophenols are surface-enhanced resonance Raman spectroscopy (Ni et al. 

De Beer TRM, Vergote GJ, Baeyens WRG, et al. Development and validation of a direct, nondestructive quantitative method for medroxyprogesterone acetate in a pharmaceutical suspension using FT-Raman spectroscopy. Eur J Pharm Sci 2004 23(4-5) 355-362. 

To validate the model deduced from the results of these Raman spectroscopy experiments detailed investigations of the Mg/PTCDA system by means of other methods that are highly sensitive with respect to the changes of the chemical environment and charge redistribution such as photoemission spectroscopy were performed [2]. 

Spectroscopic methods can provide fast, non-destructive analytical measurements that can replace conventional analytical methods in many cases. The non-destructive nature of optical measurements makes them very attractive for stability testing. In the future, spectroscopic methods will be increasingly used for pharmaceutical stability analysis. This chapter will focus on quantitative analysis of pharmaceutical products. The second section of the chapter will provide an overview of basic vibrational spectroscopy and modern spectroscopic technology. The third section of this chapter is an introduction to multivariate analysis (MVA) and chemometrics. MVA is essential for the quantitative analysis of NIR and in many cases Raman spectral data. Growth in MVA has been aided by the availability of high quality software and powerful personal computers. Section 11.4 is a review of the qualification of NIR and Raman spectrometers. The criteria for NIR and Raman equipment qualification are described in USP chapters <1119> and < 1120>. The relevant highlights of the new USP chapter on analytical instrument qualification <1058> are also covered. Section 11.5 is a discussion of method validation for quantitative analytical methods based on multivariate statistics. Based on the USP chapter for NIR <1119>, the discussion of method validation for chemometric-based methods is also appropriate for Raman spectroscopy. The criteria for these MVA-based methods are the same as traditional analytical methods accuracy, precision, linearity, specificity, and robustness however, the ways they are described and evaluated can be different. 

This chapter reviews the use of spectroscopic methods for the quantitative analysis of pharmaceutical products. In recent years, there has been great progress made in the use of techniques such as NIR and Raman for real world pharmaceutical problems. USP chapters for NIR and Raman spectroscopy outline the requirements for equipment qualification and method validation. Because spectroscopic methods for quantitative analysis often involve the use of MVA and chemometrics, the approaches for method validation are somewhat different than that for traditional chromatographic methods. 

Everall et al. [49] were interested in obtaining a simple, rapid method for determining the density of PET films and pellets. Although Raman spectroscopy had been shown to correlate with density, they found that the existing correlations were only valid when the samples consisted either only of unoriented samples or only of oriented samples, not when a combination of both types were present. They used a chemometrics approach to obtain a good correlation with density. Using four factors associated with the 1730-, 1096-, 996-, and 860-cm bands, they were able to predict the density of polymer pellets or chips and oriented films with a standard error of prediction of 0.0023 g/cm. Everall et al. [50] used a variation of this technique coupled to a confocal Raman spectrometer to... 

In the present project we have investigated whether surface enhanced resonance Raman spectroscopy (SERRS) could be used to study various types of DNA-chromophore interactions. In this technique, a silver colloid is added to the solution containing the molecule to be studied [34]. First, we had to test whether the complex, between dye and DNA, would remain unaltered upon adsorption to the silver colloid surface which is a necessary condition for a valid application of the method. A second system specific question concerns the useful information that can be extracted from frequency shifts and relative intensity alterations in the vibrational spectrum of the DNA-bound chromophore [35-37]. 

There are several site specific experimental techniques such as solids state NMR, EXAFS and Raman spectroscopy that can give additional structural information to be compared directly with simulation results and thus are able to provide further validations. For example, NMR results not only provide Qn distributions but also how the Qn species are linked together through double or multi-quantum experiments. This kind of site specific experimental methods is an additional opportunity for detailed structure comparison and validation. 

Trichloroacetic acid (TCA) protein precipitation followed by ultracentrifiigation was successful in removing proteins from HA, rapid and does not interfere with the identification of key Raman biomarker bands associated with HA. This method has been validated against commercially-available protein removal kits and found to provide superior protein-removal efficiency.(22) It should be noted that the TCA protocol dilutes HA in ASF by a factor of two. Although microscope images and Raman spectroscopy show crystalline TCA in the center of the dried droplet deposit, Raman spectra show that some TCA is still contained in the outer HA-rich rings. A broad TCA band is observed between 830-860 cm and other bands are found at 945 cm and 1365 cm. With the exception of the 945 cm band, TCA bands do not overlap with HA Raman bands and are not sources of interference. 

The use of IR and Raman spectroscopy as complementary analytical techniques (to other physical and theoretical methods) is unlimited, especially in the case of characterizations. Also, a major use of theoretical calculations is the prediction and validation of experimental (including IR and Raman) spectroscopic data, and some applications have been reported earlier. Thus, in the surface binding of DMMP, DIMP, DFP, and Sarin to silica, a DFT comparison with the experimental IR shifts showed that the theoretically-modelled adsorption sites are similar to those found by experiment, and in the case of Cyclophosphamide, the IR and Raman spectra were assigned from DFT calculations, also a homologous series of aminobisphosphonates were studied and characterized using IR and NMR spectroscopy. ... 

A promising recent development in the study of nitrenium ions has been the introduction of time-resolved vibrational spectroscopy for their characterization. These methods are based on pulsed laser photolysis. However, they employ either time resolved IR (TRIR) or time-resolved resonance Raman (TRRR) spectroscopy as the mode of detection. While these detection techniques are inherently less sensitive than UV-vis absorption, they provide more detailed and readily interpretable spectral information. In fact, it is possible to directly calculate these spectra using relatively fast and inexpensive DFT and MP2 methods. Thus, spectra derived from experiment can be used to validate (or falsify) various computational treatments of nitrenium ion stmctures and reactivity. In contrast, UV-vis spectra do not lend themselves to detailed structural analysis and, moreover, calculating these spectra from first principles is still expensive and highly approximate. 

Tt is well-known that Werner determined the structure of a number of metal complexes by skillfully combining his famous coordination theory with chemical methods (30). Modern physico-chemical methods such as x-ray diffraction and infrared spectroscopy, used in the study of Werner complexes, have paralleled the development of these techniques. The results of these investigations have not only confirmed the validity of Werner s coordination theory but have also provided more detailed structural and bonding information. In early 1932, Damaschun (13) measured the Raman spectra of seven complex ions, such as [Cu(NH3)4]" and [Zn(CN)4j and these may be the first vibrational spectra ever obtained for Werner complexes. In these early days, vibrational spectra were mainly observed as Raman spectra because they were technically much easier to obtain than infrared spectra. In 1939, Wilson 35, 36) developed a new theory, the GF method," which enabled him to analyze the normal vibrations of complex molecules. This theoretical revolution, coupled with rapid developments of commercial infrared and Raman instruments after World War II, ushered in the most fruitful period in the history of vibrational studies of inorganic and coordination compounds. 

The properties of the transmission Raman geometry are well suited to the requirements of pharmaceutical production lines, thus underlining the potential of this method to displace existing NIR absorption spectroscopy in applications where a higher chemical specificity is required. However, further studies are required to establish the technique s sensitivity limits and to validate its potential. 

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