title: The determination of a potential impurity in Thalidomide drug substance and product by HPLC with indirect UV detection.
abstract: Thalidomide molecule, a synthetic derivative of glutamine, can undergo hydrolysis at physiologic pH to form glutamine. Additionally, L-glutamine is one of the starting materials in the synthesis of Thalidomide drug substance. The current USP method for testing glutamine is thin-layer chromatography (TLC) with ninhydrin spray visualization. A more quantitative and automated high performance liquid chromatography (HPLC) method utilizing indirect ultraviolet (UV) detection was developed and validated for the determination of the non-UV absorbing glutamine in Thalidomide drug substance and product. The HPLC mobile phases consisted of phosphoric acid, 2-naphthalenesulfonate sodium and methanol. 2-Naphthalenesulfonate was used as a UV detection probe for glutamine. A segmented isocratic elution program was used to elute glutamine and Thalidomide, respectively. The method was found to be specific for glutamine. The linearity was 0.05-1.25% glutamine with respect to a nominal concentration of 8 mg ml(-1) Thalidomide sample. The limits of detection and quantitation were found to be 0.03 and 0.05% glutamine, respectively. The injection precision was 2.7% for area responses and 0.2% for the retention times. The recovery of glutamine at three concentration levels was found to be 100.8+/-2.8% from placebo and 99.2+/-5.8% from spiked Thalidomide drug substances. This newly developed HPLC method was used to determine glutamine in Thalidomide drug substances and products. The results from HPLC were in agreement with those from TLC. Therefore, the method developed is a suitable alternative to the current USP TLC procedure. Additionally, the method offers the advantage of being quantitative and automated.
authors.affiliation: Celgene Corporation, 7 Powder Horn Drive, Warren, NJ 07059, USA. jli@celgene.com
authors.forename: Jingyi
authors.initials: J
authors.lastname: Li
authors.forename: Markian S
authors.initials: MS
authors.lastname: Jaworsky
authors.forename: David I
authors.initials: DI
authors.lastname: Stirling
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004340:Drug Contamination; D005973:Glutamine / Q000032:analysis; D009282:Naphthalenesulfonates / Q000737:chemistry; D015203:Reproducibility of Results; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet; D013792:Thalidomide / Q000032:analysis*
chemical_list: D009282:Naphthalenesulfonates; D012996:Solutions; D005973:Glutamine; D013792:Thalidomide