Identification of Impurity in Blood M. Marconi M. Gara R. Dawe Travenol Laboratories, Inc. 6301 Lincoln Avenue Morton Grove, Illinois 60053
Figure 1. Structure of adenine. Molecular weight = 1 3 5
T h e purine base adenine (Figure 1) is used as a fortifier to prolong the shelf life of blood in blood storage bags (1). Addition of adenine to blood storage solutions increases blood shelf life from 21 to 35 days. Since the active ingredients come into direct contact with the blood, this manufacturing of raw material must meet the rigid specification of containing no more than 0.1% purine impurities. Such impurities may interfere with the known key roles of adenine in biological systems. To ensure t h a t adenine meets these requirements, a high performance liquid chromatography (HPLC) method with a strong cationexchange column and a phosphate buffer mobile phase was developed in our laboratories. All incoming raw materials are routinely assayed by this method. Several lots of adenine displayed a peak representing an unknown impurity t h a t was estimated to be present at concentration levels up to 0.1%. The retention time of adenine under the specified assay conditions is approximately 5 minutes, while the unknown impurity had a retention time of 45 minutes. Several possible purine, pyrimidine, and nucleoside impurities were chromatographed in an effort to identify the impurity by matching retention times. This proved to be unsuccessful. Hence, our problem was
one of isolation and identification of the unknown impurity. Specifically, our task was to determine whether the impurity was a purine.
Isolation of Impurity Initial a t t e m p t s focused on collecting the impurity as it eluted from the H P L C column and further study of the separated substance. However, it was a tedious task to acquire sufficient material for spectroscopic examination. Furthermore, removal of the impurity from the phosphate buffer mobile phase was problematic. Accordingly, a reverse phase H P L C separation with a C-18 octadecylsilane column and sodium heptane sulfonate as an ion pairing reagent was concurrently developed for the adenine raw material and the impurity. T h e impurity again had a longer retention time than adenine. Because of the chromatographic characteristics on both the ion-exchange and on the reverse phase columns, it was assumed that the impurity was more hydrophobic than adenine itself. With the above idea in mind, a solvent partitioning was attempted to selectively concentrate the impurity. T h e adenine raw material was saturated in an alkaline aqueous solution that was then extracted with chloroform. T h e chloroform layer was backwashed with basified water to remove adenine,
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and the chloroform layer was examined by H P L C to ascertain whether the impurity peak was present. Indeed, the impurity was preferentially extracted into the chloroform with only a trace of adenine.
GC and GC/MS Analysis T h e silyl derivatives of both the raw material and the chloroform extract described above were analyzed by gas chromatography (GC) with a 3-ft 3% OV-1 column. T h e trimethylsilyl (TMS) derivatives of adenine and associated purines were prepared by reacting the dry raw material with a mixture of jV,0-bis(trimethylsilyl)fluoroacetamide (BSTFA) in acetonitrile (2). Following derivatization, a gas chromatogram of the raw material was dominated by the bis-silyl derivative of adenine with several other minor components. Small amounts of mono- and tris-silylated adenine also were observed eluting immediately prior to and after the bis(silyl) derivative, respectively. T h e chloroform extract of the raw material after derivatization displayed only one prominent peak. Both samples were examined by combined gas chromatography/mass spectrometry (GC/MS). T h e mass spectrum of the T M S derivative of the impurity is shown in Figure 2. In addition, it was noted that the impurity extracted with CHCL also could be 0003-2700/79/A351-1124$01.00/0 © 1979 American Chemical Society
The Analytical Approach Edited by Claude
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Figure 2. Mass spectrum of TMS derivative of isolated impurity
Figure 3. Mass spectrum of underivatized isolated impurity
chromatographed on the same GC column without derivatization. This peak also was examined by GC/MS and its spectrum is shown in Figure 3. The mass spectrum of bis(trimethylsilyl)adenine is shown in Figure 4. A molecular ion is observed at m/e 279; however, the base peak is 264, which represents a loss of 15 mass units from the molecular ion. This loss is characteristic of silyl derivatives of purines (3). T h e mass spectrum of the T M S derivative of the impurity also shows a prominent M — 15 ion (assuming m/e 283 is the molecular ion), in addition, other characteristic ions of silylated purine include M — 15 — HCN at 241. Not characteristic of pur i n e - T M S spectra is the small ion at m/e 77 that is usually present in monosubstituted phenyl derivatives. T h e spectrum of the underivatized impurity shown in Figure 3 indicates the presence of a molecular ion at m/e 211 or 72 mass units less than the silyl derivative indicating one derivatizable hydrogen. Adenine itself predominantly forms a bis(silyl) derivative with silyl groups at 6-N and N-9. Steric hindrance retards derivatization of both 6-N hydrogens. The multiple losses of 27 (HCN), i.e., m/e 211 — 184, 184 > 157 are also indicative of a purine structure. Again, the distinct presence of the m/e 77 ion in this spectrum seemed to be indicating a
ANALYTICAL CHEMISTRY, VOL. 5 1 , NO. 11, SEPTEMBER 1979 · 1125 A
phenyl group. T h e loss of 42 (NH 2 CN) from 184 -* 142 is indicative of a purine bearing an N H 2 substitution. T h e difference in molecular weight of 76 between the impurity and adenine (MW = 135) also strongly suggests phenyl substitution at either the 6-N or N-9 position. T h e use of ion-plotting techniques (mass chromatogram) permitted the verification of the presence of the silylated impurity at very low concentrations in the silylated raw material.
Infrared and N M R S p e c t r a
More of the impurity was isolated from the raw material via the chloroform extraction technique and then subjected to infrared (IR) and Fourier transform proton nuclear magnetic resonance (NMR) spectroscopy. T h e IR spectrum of the impurity in a KBr disk was similar to, but distinct from, a file spectrum for 6-(7V-phenyl)adenine (4). T h e use of Fourier transform N M R spectroscopy allowed the acquisition of a relatively clean spectrum of the isolated impurity in deuterated chloroform (Figure 5). T h e peaks downfield, at 8.37 and 8.23 ppm, are due to the purine ring hydrogens at C-2 and C-8. T h e peak at 7.6 p p m (integrating for 5 protons) represents a monosubstituted benzene ring. T h e broad signal at 4.4 ppm (integrating for about 2 protons) is probably due to a free amino group, because the peak disappeared after deuterium exchange with D2O. All other peaks present in the spectrum are due to solvent impurities. Hence, the N M R data support the hypothesis of a phenylat-
Figure 4. Mass spectrum of bis(trimethylsilyl)adenine
Figure 5. FT proton NMR spectrum of isolated impurity
Wavelength (Microns)
Wavenumber (CM-1)
Figure 6. Infrared spectrum of synthesized 9-phenyladenine ANALYTICAL CHEMISTRY, VOL. 5 1 , NO. 11, SEPTEMBER 1979 · 1129 A
