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J Pharm Biomed Anal. 2013 Oct;84:41-7. doi: 10.1016/j.jpba.2013.05.033. Epub 2013 Jun 13.

Selective determination of potential impurities in an active pharmaceutical ingredient using HPLC-SPE-HPLC.

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Formulation Research, Pharmaceutical Science & Technology, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, Japan.


The present paper describes the selective determination of two synthetic intermediates (2,4-dichloro-6,7-dimethoxyquinazoline (IMP-1) and its derivative (IMP-2) as potential impurities in the active pharmaceutical ingredient (API)-A using two-dimensional high-performance liquid chromatography (HPLC) hyphenated via on-line solid-phase extraction (SPE) (HPLC-SPE-HPLC). Two synthetic intermediates that are potential impurities in API-A were concentrated on-line on two Shimadzu MAYI-ODS SPE columns (10 mm×4.6 mm I.D.) after heartcutting in 1st dimension HPLC (1st HPLC) using a Shiseido CAPCELL PAK ACR C18 column (250 mm × 10.0 mm I.D.). Each analyte retained on these SPE columns was transferred to 2nd dimension HPLC (2nd HPLC) with a Shiseido CAPCELL PAK MG-II column (150 mm × 3.0 mm I.D.) for further separation and was subsequently detected with high sensitivity UV. The HPLC-SPE-HPLC system achieved a stepwise downsizing in HPLC. The method was validated and found to be accurate and precise with a linear range of 0.25-250 ppm of each intermediate in API-A with respect to a 500 μL injection of 40 mg/mL of API-A in dimethyl sulfoxide. The method was successfully applied for the determination of these impurities in API batches, and the results demonstrated the usefulness of HPLC-SPE-HPLC for the selective determination of trace impurities in APIs.


Impurity; Multi-dimensional HPLC; Selective determination; Solid-phase extraction; Synthetic intermediate

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