Andrew P. Cheung

New stability-indicating high performance liquid chromatography assay and proposed hydrolytic pathways of chlorhexidine

Chlorhexidine (CHD) is an antiseptic agent widely used in mouth rinses and ophthalmic solutions. Its assay in these solutions has been commonly performed with high performance liquid chromatography (HPLC). Published HPLC assays were unable to detect the many possible hydrolysis products of CHD, with the exception of p-chloroaniline. This raises concerns regarding the stability-indicating capabilities of the HPLC assays. In addition, the reliability of these assays was affected by the irreversible absorption of CHD on silica-based reversed phase (RP) columns. Special attention must be paid to the mobile phase or sample matrices in order to obtain reliable assay results. This paper presents a polymer-based RP-HPLC method that is stability-indicating. The method detected six UV-absorbing hydrolysis products of CHD. The identities of the products, which were elucidated by UV and mass spectral analyses, are consistent with the proposed hydrolytic pathways of CHD. Quantitation problems observed with silica-based RP-HPLC assays do not arise with the new method. Preliminary data indicate that the new HPLC method can be developed into a reliable, stability-indicating assay for CHD in ophthalmic solutions.

Stability-indicating LC assay of and impurity identification in homoharringtonine samples ☆

Homoharringtonine (HHT) is a potent myelosuppressive agent and has antitumor activity. Recent studies suggest that it inhibits tumor growth by inducing apoptosis. HHT is an ester of the alkaloid cephalotaxine. It is isolated from genus Cephalotaxus. At least ten HHT analogs have been identified from cephalotaxus extracts. High performance liquid chromatography (HPLC) separations of the cephalotaxine alkaloids in plant extracts have been reported, they have not been validated as specific and stability-indicating for HHT. Due to the complexity of the alkaloid extracts, it is conceivable that additional analogs may still be unresolved from HHT. This paper presents an improved and validated HPLC assay for HHT. The assay is stability-indicating, precise (R.S.D. < 1%), linear (r2 = 0.9999), and accurate (error < 1%). The assay reveals three congeners present as impurities in HHT samples. Two are new and have not been previously reported. Identities of the impurities and forced decomposition products, elucidated with their HPLC retention and spectral data, are also presented.

Application of HPLC and CZE to the analysis of polyoxometalates

Polyoxometalates (POM) are polymers of transition metal oxides. They are widely used as analytical reagents and reaction catalysts. Some have anti-viral properties and are being investigated as anti-HIV agents. Due to solubility and stability limitations, separation methods for POM are rare in the literatures. This paper presents a HPLC and a CZE method for the analysis of sodium tungstate, its equilibrium products and isopolyanions. The methods are simple and sensitive, and can be used to monitor the purity, stability and solution equilibria of POM.

Precision in capillary electrophoresis with respect to quantitative analysis of suramin

Suramin is an important anti-tumor and anti-viral chemotherapeutic agent. We have previously presented a capillary electrophoresis (CE) method for its quantitative analysis, where its quantitation was linear over three orders of magnitude, with good precision (1.8%) and accuracy. The constantly varying electroosmotic properties of the capillary due to various causes such as analyte adsorption to the inner wall, affect the migration times of analytes during consecutive electrophoresis runs. This results in progressive changes in analyte peak areas, causing less desirable or unacceptable CE assay precision. This paper illustrates a strategy to overcome the problem of assay reproducibility by using an internal standard whose migration time is short and close to that of the analyte so that the relative change of migration time is minimized. Assay precisions as good as 0.3% were observed in these experiments. These results are in agreement with the theoretical basis of experimental capillary electrophoresis.

Compatibility and stability of bryostatin 1 in infusion devices

Bryostatin 1 is currently in phase II clinical trial sponsored by the National Cancer Institute as an anticancer chemotherapeutic agent. Bryostatin 1 for injection was supplied in a dual pack containing a drug vial and a diluent vial and was manufactured by Ben Venue Laboratories, Inc (Bedford, OH). The stability and compatibility of the bryostatin 1-PET formulation, diluted to 1 and 10 ug/mL in saline and benzyl alcohol preserved saline, with polypropylene (PP) and polyvinyl chloride (PVC) bags at room temperature (27°C) were studied. All experiments were conducted in triplicate and analyses were performed using a validated, stability-indicating, high performance liquid chromatography (HPLC) assay.Bryostatin 1 solutions were compatible with PP bags. At both concentrations and with both salines, the bryostatin content remained unchanged during the 28-day storage period, benzyl alcohol concentration in the preserved saline solutions also remained relatively constant. In PVC bags, however, a decrease in bryostatin 1 concentrations without generation of decomposition products was observed at both dilutions and with both salines during the 28-day storage. A decrease in benzyl alcohol concentration in the preserved saline was also observed. While no diethylhexylphthalate (DEHP) leakage into the solution was observed in PP bags, DEHP leakage in PVC infusion bags was observed on day 2 of storage which increased with storage time and leveled off on day 6. The amount of DEHP leached into drug solution is dependent on the drug concentration. This study suggests bryostatin-PET formulation diluted with preserved saline can be used for long-term (4 week) intravenous administration using PP infusion bags, but not with PVC bags.

Chiral separation of glycyrrhetinic acid by highperformance liquid chromatography

An HPLC separation of the 18 alpha and 18 beta diastereomers of glycyrrhetinic acid (GA) has been developed. The separation was achieved with a chiral column (Merck ChiraSpher, Darmstadt, Germany) and a methanol-water (65:35, v/v) mobile phase. The separation is excellent, having a resolution factor of 2.3. The HPLC method is efficient and accurate for the optical purity determination of 18 alpha- and 18 beta-GA samples. The run time is less than 30 min. Quantitation is linear with a lower detection limit of 0.2% for one isomer in the presence of the other. Addition of 0.2% acetic acid to the water in the mobile phase improved the ruggedness of the system without affecting the resolution of 18 alpha- and 18 beta-GA. This modification also allowed partial separation of 18 alpha- and 18 beta-glycyrrhizin, precursors of the GA isomers.

Problems affecting the liquid chromatographic quantitation of chlorhexidine digluconate in ophthalmic solutions

A liquid chromatogrpahic (LC) assay was recommended by the Food and Drug Administration of the United States for the uptake and release study of chlorhexidine digluconate (CHDG) in ophthalmic solutions by contact lenses. The results from this and other reversed-phase LC assays of CHDG were inaccurate when the working standard and sample solution matrices were different. The error was caused by binding of the analyte onto the container surface and LC column packings. The loss of chlorhexidine due to binding was dependent upon, and very sensitive to, the counter ions in the sample solutions. Relative to water solutions of CHDG, solutions containing chloride, thiosulphate and edetate reduced the loss of the analyte, while solutions containing borate enhanced the loss. To assay CHDG reliably with reversed-phase LC, the media of the working standard and the sample solutions should be closely matched.

Stability-indicating HPLC assay and solution stability of a new diaziridinyl benzoquinone

RH1, 3-methyl-6-hydroxymethyl-2,5-diaziridinyl-1,4-benzoquinone, is a NQO1 (NAD(P)H quinone oxidoreductase) directed anti-tumor agent. It is designed as a water soluble analog of MeDZQ (3,6-dimethyl-2,5-diaziridinyl-1,4-benzoquinone) and is a drug candidate for clinical evaluation. A HPLC assay has been developed for its analysis. The assay is sensitive (ldl<0.2 ng), precise (rsd<1%), linear (r(2)=0.9997), accurate (error<0.6%), and stability-indicating. Using the developed assay, aqueous stability of RH1 has been evaluated. Both aziridine rings in MeDZQ are known to be easily hydrolyzable in aqueous solutions, however, hydrolysis of the second aziridine ring in RH1 appears inhibited.

A new sensitive HPLC assay for methoxyamine and its analogs.

Methoxyamine (MOA) and its analogs are polymerization regulators, building blocks and intermediates for agrichemicals and pharmaceuticals. MOA induces mutagenesis of nucleic acids and has been considered for anti-cancer and anti-virus therapy. It has been studied as a DNA repair modifier in anti-cancer therapy. HPLC procedures available in the literature for MOA are all based on electrochemical detection, which is not commonly available. This paper describes the development and validation of a HPLC assay with UV detection for MOA and its analogs. The analytes are first reacted with o-phthalaldehyde to form an oxime derivative before chromatography with an ODS column. Detection is achieved by UV at 254 nm. The chromatography resolves MOA from its decomposition products and analogs. The assay is reproducible (R.S.D. < 0.8%), linear (r(2) = 0.9997), and accurate (error < 1%). The method is sensitive and has a lower detection limit of 5 pmol (0.4 ng of MOA.HCl), which is comparable to that of electrochemical detection.

Quantitative capillary electrophoresis and its application to the polyanionic quinobene

Factors affecting the accuracy of capillary electrophoresis (CE) assays in general are discussed. Methods to improve the reproducibility and reliability of these assays are suggested. The improvements are demonstrated by developed CE assays for quinobene and suramin. The assays were reproducible (RSD < 2%), accurate (error < 2%), and linear over a concentration range of 1-800 micrograms ml-1 (r2 = 0.999).