Technical Information
Application Brief AB-055
The analysis of active pharmaceutical ingredients (APIs) in thesolid state is critically important in drug development andquality assurance, because many drugs are dispensed assolids. Indeed, the APIs may exist in different polymorphiccrystalline or amorphous states, as hydrates or solavates, oreven as salts with various counterions. When a drug substanceis formulated, knowing and controlling the composition andstate of the API is necessary to produce an effective product.
Additionally, there are legal implications related to patentclaims and intellectual property that can translate into millions Figure 1. Photomicrographs of Furosemide recrystallized from (left) of dollars in revenue if a novel API is properly characterized.
ethanol and (right) n-butanol.
The technical challenge lies in the inability of a single analytical technique to fully characterize the many different forms of the many APIs of interest. From Ethanol From n-Butanol This Application Note discusses the synergy of combining light microscopy, infrared (IR) microspectroscopy and X-Ray powder diffraction (XRPD) for characterizing the solid state forms of two chemically and structurally diverse APIs. The results demonstrate the need for performing analyses on both the visual and the molecular levels, such that a completeunderstanding of the API is obtained.
Furosemide (CAS# 54-31-9) and Dirithromycin (CAS# 62013-04-1) were purchased from Sigma-Aldrich Chemical Co. and used Wavenumber (cm-1) without further purification. All solvents used for recrystallization Figure 2. Infrared diamond ATR spectra of two forms of Furosemide were anhydrous and 99% pure. Various crystalline forms of crystallized from two different solvents. The green asterisks highlight Furosemide and solvates of Dirithromycin were prepared major differences in the spectra.
according to the literature references1-3. Infrared spectra of thesamples were obtained in diamond attenuated total reflection (ATR) mode using a SensIR Technologies IlluminatIR FT-IR microspectrometer accessory attached to an Olympus BX-51 microscope. XRPD patterns were obtained using a Phillips multi- From n-Butanol purpose diffractometer set up with Bragg-Brentano optics and scanning from 5-50 2 Theta.
Results and Discussion
Furosemide: Furosemide is a diuretic and antihypertensive drug that crystallizes as different solid state forms, depending on the boiling point of the recrystallization solvent. In fact, three distinct polymorphs, two solvates and an amorphous form have been reported in the literature . In the current study, one polymorph of Furosemide was produced by rapid recrystallizationfrom a hot, saturated ethanol solution, while another was prepared by slow recrystallization from saturated n-butanol. As shown in A simple rotation of the microscope nosepiece allowed the IR Figure 1, both polymorphs crystallized as needles. Those diamond ATR spectra to be obtained. In the spectra in Figure 2, formed from ethanol were smaller on average, presumably due distinct differences in the relative intensities and positions of to the relatively rapid crystal seed formation and growth.
several absorption bands indicate the presence of two Overall, both preparations appeared similar under the polymorphs. This is particularly evident in the O-H and N-H microscope, and any differences in crystal structure related to stretching region from 3400-3200 cm-1, where the spectral polymorphism were not evident. band characteristics indicate differences in the hydrogen bonding networks of the two crystal structures. Polymorphism was confirmed by XRPD analysis of the two Furosemidepreparations. As shown in Figure 3, the crystals grown fromethanol and n-butanol exhibit very different diffraction patterns.
Indeed, this experiment shows that confirmatory analyticaltechniques like IR and XRPD are essential for detecting andcharacterizing API polymorphs when visually similar crystalsare formed.
Dirithromycin: Dirithromycin is a semisynthetic antibiotic thatforms two anhydrous polymorphs, an amorphous solid, and at Figure 4. Polarized light photomicrographs of Dirithromycin (left) asreceived and (right) recrystallized from acetone.
least nine stoichiometric solvates2,3. In this experiment,Dirithromycin was studied as received (a.k.a., the "native"form), and as solvates of acetone and 2-propanol. Unlike the Furosemide example, the light microscope images (see Figure 4) of the native Dirithromycin and the acetone preparation clearly indicate two different solid state habits. The distinct XRPD patterns in Figure 5 support the conclusion of different crystalline structures. Indeed, the IR spectra in Figure 6 suggest that the acetone preparation is a solvate, because the spectra are similar except for an additional C=O band at 1734.5 cm-1 in the acetone preparation due to the small acetone solvent molecule being held within the larger API molecule .
As noted by Stephenson, et al.3, the acetone and 2-propanol solvates of Dirithromycin exhibit nearly identical X-Ray diffraction patterns, rendering them indistinguishable by XRPD.
In this experiment, the 2-propanol solution always produced an Figure 5. XPRD patterns of the three Dirithromycin preparations, indicating amorphous solid, and no characteristic X-Ray diffraction two different crystal structures for the native form and acetone solvate, and pattern of a crystal was obtained (see Figure 5). However, the an amorphous 2-propanol solvate.
IR spectra of two solvent preparations are distinguishable fromeach other and from the native form, as shown in Figure 6. In particular, solvation by 2-propanol shifts the C=O band in the Dirithromycin spectrum from 1708 to 1731 cm-1, and the free O-H band at 3539 cm-1 (in the native form) becomes hydrogen- bonded. In addition, an O-H band from the alcohol solvent is newly apparent in the solvate spectrum, proving that IR microspectroscopy can provide important chemical details thatX-Ray analysis cannot. Ultimately, this experiment shows how light microcopy and XRPD can often distinguish different API forms, but the molecular information provided by IR is essential when solvates are produced.
1. Matsuda, Yoshihisa and Etsuko Tatsumi, International Wavenumber (cm-1) Journal of Pharmaceutics, 60, pp. 11-26 (1990) Figure 6. Infrared diamond ATR spectra of Dirithromycin in unsolvated and 2. Byrn, Stephen R., Ralph R. Pfeiffer and Joseph G. Stowell, solvated forms. The observed differences are primarily due to the presence Solid State Chemistry of Drugs, 2nd Ed.,West Lafayette: SSCI, of solvent molecules.
Inc. (1999)3. Stephenson, Gregory A., et al., Journal of the American Chemical Society, 116, pp. 5766-5773 (1994) Figure 3. ATR spectra of 108 ng of acrylic polymer deposited from 10.0 mLof 13.4 ppm solution onto 1 and 9 reflection diamond / zinc selenideDuraDisks.
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