A Sensitive LC-MS/MS Method for the Simultaneous Determination of Two Thia-Analogous Indirubin N-Glycosides and Indirubin-3′-Monoxime in Plasma and Cell Culture Medium

Indirubin was identified as an active component of Danggui Longhui Wan, an herbal mixture used in traditional Chinese medicine, and showed anticancer activity in clinical trials in patients with chronic leukemia. Investigations on the mechanisms of antitumor action of indirubins have mainly focused on the indirubin derivative indirubin-3 '-monoxime (I3M). Meanwhile, antiproliferative and cytotoxic properties on cancer cells have also been demonstrated for several synthetic indirubin N-glycosides. In the present study, we demonstrate cytotoxic activity of the thia-analogous indirubin N-glycosides KD87 (3-[3 '-oxo-benzo[b]thiophen-2 '-(Z)-ylidene]-1-(beta-d-glucopyranosyl)-oxindole) and KD85 (3-[3 '-oxo-benzo[b]thiophen-2 '-(Z)-ylidene]-1-(beta-d-mannopyranosyl)-oxindole) against melanoma and squamous cell carcinoma cells as well as lung cancer and glioblastoma cells. The advanced state of preclinical studies on the effects of indirubins conducted to date underscores the need for pharmacokinetic data from cellular, animal, and human studies for which reliable quantification is required. Therefore, a sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous measurement of KD87, KD85, and I3M in plasma and cell culture medium. Experimental conditions for sample preparation were optimized for human plasma protein precipitation and liquid-liquid extraction from plasma and cell culture medium. The methods were successfully validated in accordance with the U.S. Food and Drug Administration Bioanalytical Method Validation and evaluated for selectivity, sensitivity, matrix effect, recovery, carryover, calibration curve linearity, accuracy, precision, and stability. The applicability of the methods was demonstrated by the determination of KD87 in mouse plasma after prior intraperitoneal administration to mice.

Automated summary

Indirubin has been identified as an active component in a traditional Chinese herbal mixture and has shown anticancer activity in patients with chronic leukemia. Multiple studies have focused on the mechanisms of its antitumor action, particularly the derivative indirubin-3'-monoxime (I3M). Additionally, synthetic indirubin N-glycosides have demonstrated antiproliferative and cytotoxic properties in various cancer cells. A sensitive LC-MS/MS method was developed and validated for quantification of indirubin N-glycosides KD87, KD85, and I3M in plasma and cell culture medium. The applicability of the method was demonstrated in a mouse plasma study.