Euphemia Wang

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.

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.

LC/MS characterization of impurities and degradation products of a potent antitumor peptidic dimer, CU201.

Compound CU201 [SUIM-(d-Arg-Arg-Pro-Hyp-Gly-Igl-Ser-d-Igl-Oic-Arg)(2), where SUIM=suberimidyl; Hyp=trans-4-hydroxyproline; Igl=alpha-(2-indanyl)-glycine; Oic=octahydroindole-2-carboxylic acid], is a dimeric analog of the potent bradykinin antagonist peptide B9430. It blocks the G(alphaq,11) signal of the heterotrimeric G proteins, stimulates c-Jun kinases, and induces apoptosis in lung cancer cells with neuroendocrine features. CU201 shows potent inhibition for small-cell lung cancer cells in vitro (ED(50)=0.15microM), as well as for small-cell lung cancer SHP-77 tumor growth in vivo. An HPLC method was developed, as part of a study supported by the National Cancer Institutes (NCIs) Rapid Access to Interventional Development (RAID) program, to assess the purity and stability of CU201. Impurities and degradation products were characterized by LC/MS. The identity of a major impurity, with 1 mass unit different from CU201, was confirmed by high resolution LC/MS and the investigation of model compounds. Susceptible linkages in the peptide chains were revealed by the degradation study.

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.

Characterization, HPLC method development and impurity identification for 3,4,3-LI(1,2-HOPO), a potent actinide chelator for radionuclide decorporation.

3,4,3-LI(1,2-HOPO), 1,5,10,14-tetra(1-hydroxy-2-pyridon-6-oyl)-1,5,10,14-tetraazatetradecane), is a potent octadentate chelator of actinides. It is being developed as a decorporation treatment for internal contamination with radionuclides. Conventional HPLC methods exhibited speciation peaks and bridging, likely attributable to the agents complexation with residual metallic ions in the HPLC system. Derivatization of the target ligand in situ with Fe(III) chloride, however, provided a single homogeneous iron-complex that can readily be detected and analyzed by HPLC. The HPLC method used an Agilent Eclipse XDB-C18 column (150 mm × 4.6mm, 5 μm) at 25°C with UV detection at 280 nm. A gradient elution, with acetonitrile (11% to 100%)/buffer mobile phase, was developed for impurity profiling. The buffer consisted of 0.02% formic acid and 10mM ammonium formate at pH 4.6. An Agilent 1200 LC-6530 Q-TOF/MS system was employed to characterize the [Fe(III)-3,4,3-LI(1,2-HOPO)] derivative and impurities. The proposed HPLC method was validated for specificity, linearity (concentration range 0.13-0.35 mg/mL, r = 0.9999), accuracy (recovery 98.3-103.3%), precision (RSD ≤ 1.6%) and sensitivity (LOD 0.08 μg/mL). The LC/HRMS revealed that the derivative was a complex consisting of one 3,4,3-LI(1,2-HOPO) molecule, one hydroxide ligand, and two iron atoms. Impurities were also identified with LC/HRMS. The validated HPLC method was used in shelf-life evaluation studies which showed that the API remained unchanged for one year at 25°C/60% RH.

HPLC method development, validation and impurity characterization for an antitumor Hsp90 inhibitor—PU-H71 (NSC 750424)

An HPLC method for the assay of the heat shock protein 90 inhibitor, PU-H71 (NSC 750424), has been developed and validated. The stress testing of PU-H71 was carried out in accordance with ICH guidelines Q1A (R2) under aqueous, acidic, alkaline, oxidative, thermolytic and photolytic conditions. The separation of PU-H71 from its impurities and degradation products was achieved within 50min on a Mac-Mod ACE 3 C18 column (150mm×4.6mm i.d., 3μm) with a gradient mobile phase comprising 20-95% acetonitrile in water, with 0.1% trifluroacetic acid in both phases. LC-quadrupole TOF/MS was used to obtain accurate mass data on various components as well as on their fragments for characterization of impurities and degradation products. The proposed HPLC assay method was validated for specificity, linearity (concentration range 0.1-0.3mg/mL, r≥0.9998), accuracy (recovery 99.7-101.1%), precision (intra-lab RSD≤1.39%, inter-lab RSD≤0.91%), sensitivity (LOD 0.08μg/mL), and ruggedness. The developed method was suitable for the assay and stability monitoring of PU-H71 drug substance.

High-performance liquid chromatographic analysis for a non-chromophore-containing phosphatidyl inositol analog, 1-((1-O-octadecyl-2-O-methyl-sn-glycero)phospho)-1D-3-deoxy-myo-inositol, using indirect UV detection.

Phosphatidylinositide-3-kinase (PI3 kinase) is an important constituent of growth factor regulation. It is also involved in oncogene signaling pathways. An ether-containing phosphatidyl inositol(PI) analog, OMDPI, 1-[(1-O-octadecyl-2-O-methyl-sn-glycero)-phospho]-1D-3-deoxy-myo-inositol, is a potent inhibitor of this pathway and may be clinically useful in the treatment of a variety of neoplasms. OMDPI is currently being investigated as an anti-tumor agent by the National Cancer Institute, NIH. OMDPI, a non-chromophore-containing PI analog, is not directly adaptable to the commonly used UV detection of HPLC. This paper reports the development and validation of an HPLC assay for OMDPI based on indirect UV detection, in which a UV-absorbing ion-pair reagent (the probe), protriptyline, is added to the mobile phase to induce a signal for the compound. The method is sensitive (limit of detection <5 microl of 1 microg/ml or 5 ng), precise (R.S.D.<2.5%), linear (r2=0.9995) and accurate (error<0.7%). It is superior to refractive index detection and evaporative light scattering detection in either sensitivity or linearity and does not require special equipment.

Enantiomeric separation of an aryloxyphenoxypropanoic acid by CE and LC.

A capillary electrophoresis (CE) and an high performance liquid chromatography (HPLC) chiral separation have been developed for an aryloxyphenoxypropanoic acid, 2-[4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy]propanoic acid, a new antitumor agent. The racemic mixture is analyzed, without derivatization, as the free acids. The CE assay is based on inclusion complexation with hydroxypropyl-beta-cyclodextrin. HPLC separation is achieved with a CSP column with the glycopeptide, teicoplanin, as the chiral selector. Both methods give baseline resolution to the R-and S-isomers. The methods were validated for assay and for optical purity assessment of the R-isomer. For assay, the HPLC method is precise (RSD < 0.6%), accurate (error, 0.5%) and linear (r2 = 0.9998). It is able to precisely (RSD = 0.5%) and accurately (error, 0.9%) detect 0.3-6.0% of one isomer (S) in the other (R). The CE assay is much less precise and accurate than HPLC. It is a good alternative to separate and detect the enantiomers, however.

HPLC method development, validation, and impurity characterization of a potent antitumor indenoisoquinoline, LMP776 (NSC 725776).

An HPLC method for the assay of a DNA topoisomerase inhibitor, LMP776 (NSC 725776), has been developed and validated. The stress testing of LMP776 was carried out in accordance with International Conference on Harmonization (ICH) guidelines Q1A (R2) under acidic, alkaline, oxidative, thermolytic, and photolytic conditions. The separation of LMP776 from its impurities and degradation products was achieved within 40 min on a Supelco Discovery HS F5 column (150 mm × 4.6 mm i.d., 5 μm) with a gradient mobile phase comprising 38-80% acetonitrile in water, with 0.1% trifluoroacetic acid in both phases. LC/MS was used to obtain mass data for characterization of impurities and degradation products. One major impurity was isolated through chloroform extraction and identified by NMR. The proposed HPLC assay method was validated for specificity, linearity (concentration range 0.25-0.75 mg/mL, r = 0.9999), accuracy (recovery 98.6-100.4%), precision (RSD ≤ 1.4%), and sensitivity (LOD 0.13 μg/mL). The validated method was used in the stability study of the LMP776 drug substance in conformance with the ICH Q1A (R2) guideline.