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Hydrolytic degradation products of cefdinir were studied in acidic (pH 1), neutral (pH 6), and basic (pH 9) solutions. Seven major degradation products were isolated by preparative and/or high-performance liquid chromatography and characterized by UV, IR, 1H-NMR, and mass spectra. To clarify degradation pathways in each pH solution, kinetic and product analyses during hydrolysis of cefdinir were carried out along with the followup reaction of representative degradation products. Cefdinir was shown to degrade via two major degradation routes: beta-lactam ring-opening and pH-dependent isomerizations (lactonization, epimerization at C-6 or C-7, syn-anti isomerization of N-oxime function).

Hydrolysis of cefdinir leads to pH-dependent isomerizations and beta-lactam ring-opening. Lactam ring opened gamma-lactones were produced as a mixture of four diastereoisomers based on the lactone methyl, and C-6 isomerizations in acidic to neutral solutions. Cefdinir and its 7-epimer were hydrolyzed to clarify the pathway leading to these lactones and the mechanism of C-6 epimerization with the aid of chiral separation techniques. Chiral separation using a bovine serum albumin column was employed to detect the beta-lactam ring opened products of cefdinir and its 7-epimer; the C-6 and C-7 isomerization was thereby observed; however, it was found to be pH-dependent at pH > or = 9. Optical activity detection applied to the lactones produced from cefdinir and its 7-epimer demonstrated that the corresponding peaks of these lactones were enantiomeric pairs. In addition, the smallest rate constant at pH 4 was observed for C-6 epimerization of the lactones, and it was found to proceed without deprotonation at C-6 by 1H-NMR spectroscopy. From the results of these studies, a plausible mechanism for C-6 epimerization has been proposed. Additionally, it was confirmed that two degradation pathways were involved during hydrolysis of cefdinir to the lactone.

The validation of the HPLC method used for the determination of cefdinir and its related substances is described. The developed method was specific and stability-indicating and provided a linear response with concentration. The system and method precision, expressed as relative standard deviations, were not greater than 1%, and the reproducibilities within and between laboratories were acceptable for the assay method. The procedure can quantitate related substances greater than approximately 0.05% of the principal cefdinir peak.

A high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of FK506, a new immunosuppressant, in bulk drug samples and dosage forms. Equilibration between FK506 (I) and its tautomeric compounds (II and III) was accomplished “artificially” in water-dehydrated alcohol (1∶1) employed as an extraction solvent. After reaching equilibrium, separation of I, treated as the representetive of equilibrated FK506, from its related substances was achieved by reversed-phase HPLC on a C18 column with water-isopropyl alcohol-tetrahydrofuran (5∶2∶2, v/v) as the mobile phase, and detection at 220 nm. Component I in dosage forms could be recovered satisfactorily and determined with good precision. The calibration graph was linear over the range 2–6 μg for I.

In the United States Pharmacopeia (USP), 0.1 N perchloric acid in acetic acid volumetric solution (hereafter HClO4 VS) used for non-aqueous titration has specified a water content between 0.02 and 0.05%. Preparing this titrant with such a narrow range of water content is very time consuming, precludes the use of commercially available titrants, and, consequently, prompted an investigation to try and expand the range up to 0.5%. In this study, the titrimetric results obtained using HClO4 VS containing more water were very close to those obtained using the USP specified titrants. A maximum assay difference of 0.7% in the titrations of three selected nitrogen-containing compounds, clonidine hydrochloride, dipyridamole, and adenosine were observed. The titrimetric results obtained using these titrants were also precise with RSDs of not more than 0.4%. Therefore, a wider range of water content in HClO4 VS between 0.02 and 0.5% is suggested for the USP potentiometric titration of nitrogen-containing compounds.

A rapid quantitative analysis of the sialylated N-linked oligosaccharides of recombinant erythropoietin (EPO) expressed in Chinese hamster ovary (CHO) cells has been developed. The procedure utilizes a glycoamidase (glycopeptidase F) to release all of the N-linked oligosaccharides from the native glycoprotein, followed by direct chromatographic analysis using high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection. The eight sialyloligosaccharides isolated from HPAEC were characterized by derivatizing with 2-aminopyridine followed by two-dimensional HPLC mapping of the pyridylaminated asialooligosaccharides (Tomiya et al., 1988, Anal. Biochem. 171, 73-90). Seven kinds of complex-type asialooligosaccharides were identified ranging from a biantennary structure to N-acetyllactosamine-extended tetraantennary structure. Approximately 3% of the terminal galactose residues of the oligosaccharides released from EPO were not sialylated whereas 97% contained an alpha(2-->3)-linked sialic acid. Quantitative oligosaccharide mapping of four different lots of EPO from CHO cells was performed to quantify the molar balance and distribution of the N-linked oligosaccharides. The sialyloligosaccharides were distributed with approximately 5% disialylated (single type), 20% trisialylated (six types), and 75% tetrasialylated (four types) oligosaccharides with an average molar recovery of 85% starting from 750 pmol of EPO.

Hydrolysis of cefixime in buffer solutions (pH 1-9) at 25 degrees C and a constant ionic strength of 0.3 was investigated using ion-pair reversed-phase HPLC. Hydrolysis rates followed pseudo first-order kinetics; the rate of hydrolysis of cefixime was very slow at pH 4-7, slightly faster at lower pH, and quite rapid at higher pH. In the early stages of hydrolysis, six major degradation products were isolated and identified: a beta-lactam ring-opened product and a 7-epimer (basic conditions), three lactones derived from intramolecular cyclization between the 2-carboxyl and 3-vinyl groups (acidic conditions), and an aldehyde derivative involving a 7-acyl moiety (neutral conditions). Principal degradation pathways for cefixime were found to involve initial cleavage of the beta-lactam ring.