Simultaneous determination of cations, zwitterions and neutral compounds using mixed-mode reversed-phase and cation-exchange high-performance liquid chromatography.

A novel mixed-mode reversed-phase and cation-exchange high-performance liquid chromatography (HPLC) method is described to simultaneously determine four related impurities of cations, zwitterions and neutral compounds in developmental Drug A. The commercial column is Primesep 200 containing hydrophobic alkyl chains with embedded acidic groups in H(+) form on a silica support. The mobile phase variables of acid additives, contents of acetonitrile and concentrations of potassium chloride have been thoroughly investigated to optimize the separation. The retention factors as a function of the concentrations of potassium chloride and the percentages of acetonitrile in the mobile phases are investigated to get an insight into the retention and separation mechanisms of each related impurity and Drug A. Furthermore, the elution orders of the related impurities and Drug A in an ion-pair chromatography (IPC) are compared to those in the mixed-mode HPLC to further understand the chromatographic retention behaviors of each related impurity and Drug A. The study found that the positively charged Degradant 1, Degradant 2 and Drug A were retained by both ion-exchange and reversed-phase partitioning mechanisms. RI2, a small ionic compound, was primarily retained by ion-exchange. RI4, a neutral compound, was retained through reversed-phase partitioning without ion-exchange. Moreover, the method performance characteristics of selectivity, sensitivity and accuracy have been demonstrated to be suitable to determine the related impurities in the capsules of Drug A.

Clinical pharmacokinetics of thalidomide.

Thalidomide is a racemic glutamic acid derivative approved in the US for erythema nodosum leprosum, a complication of leprosy. In addition, its use in various inflammatory and oncologic conditions is being investigated. Thalidomide interconverts between the (R)- and (S)-enantiomers in plasma, with protein binding of 55% and 65%, respectively. More than 90% of the absorbed drug is excreted in the urine and faeces within 48 hours. Thalidomide is minimally metabolised by the liver, but is spontaneously hydrolysed into numerous renally excreted products. After a single oral dose of thalidomide 200 mg (as the US-approved capsule formulation) in healthy volunteers, absorption is slow and extensive, resulting in a peak concentration (C(max)) of 1-2 mg/L at 3-4 hours after administration, absorption lag time of 30 minutes, total exposure (AUC( infinity )) of 18 mg. h/L, apparent elimination half-life of 6 hours and apparent systemic clearance of 10 L/h. Thalidomide pharmacokinetics are best described by a one-compartment model with first-order absorption and elimination. Because of the low solubility of the drug in the gastrointestinal tract, thalidomide exhibits absorption rate-limited pharmacokinetics (the 'flip-flop' phenomenon), with its elimination rate being faster than its absorption rate. The apparent elimination half-life of 6 hours therefore represents absorption, not elimination. The 'true' apparent volume of distribution was estimated to be 16L by use of the faster elimination-rate half-life. Multiple doses of thalidomide 200 mg/day over 21 days cause no change in the pharmacokinetics, with a steady-state C(max) (C(ss)(max)) of 1.2 mg/L. Simulation of 400 and 800 mg/day also shows no accumulation, with C(ss)(max) of 3.5 and 6.0 mg/L, respectively. Multiple-dose studies in cancer patients show pharmacokinetics comparable with those in healthy populations at similar dosages. Thalidomide exhibits a dose-proportional increase in AUC at doses from 50 to 400 mg. Because of the low solubility of thalidomide, C(max) is less than proportional to dose, and t(max) is prolonged with increasing dose. Age, sex and smoking have no effect on the pharmacokinetics of thalidomide, and the effect of food is minimal. Thalidomide does not alter the pharmacokinetics of oral contraceptives, and is also unlikely to interact with warfarin and grapefruit juice. Since thalidomide is mainly hydrolysed and passively excreted, its pharmacokinetics are not expected to change in patients with impaired liver or kidney function.

Clinical pharmacokinetics of thalidomide

Thalidomide is a racemic glutamic acid derivative approved in the US for erythema nodosum leprosum, a complication of leprosy. In addition, its use in various inflammatory and oncologic conditions in being investigated. Thalidomide interconverts between the (R)- and (S)-enantiomers in plasma, with protein binding of 55% and 65%, respectively. More than 90% of the absorbed drug is excreted in the urine and faeces within 48 hours. Thalidomide is minimally metabolised by the liver, but is spontaneously hydrolysed into numerous renally excreted products. After a single oral dose of thalidomide 200mg (as the US-approved capsule formulation) in healthy volunteers, absorption is slow and extensive, resulting in a peak concentration (Cmax) of 1-2mg/L at 3-4 hours after administration, absorption lag time of 30 minutes, total exposure (AUCoo) of 18mg - h/L, apparent elimination half-life of 6 hours and apparent systemic clearence of 10 L/H. Thalidomide pharmacokinetics are best described by a one-comportment model with first-order absorption and elimination. Because of the low solubility of the drug in the gastrointestinal tract, thalidomide exhibits absorption rate-limited pharmacolinetics (the 'flip-flop' phenomenon), with its elimination rate being faster than in absorption rate. The apparent elimination half-life of 6 hours therefore represents absorption, not elimination. The 'true' apparent volume of distribution was estimated to be 16L by use of the faster elimination-rate half-life. Multiple doses of thalidomide 200 mg/day over 21 days cause no change in the pharmacokinetics, with a steady-state Cmax (Cssmax) of 1.2 mg/L. Simulation of 400 and 800 mg/day also shows no accululation, with Css of 3.5 and 6.0 mg/L, respectively. Multiple-dose studies in cancer patients show pharmacokinetics comparable with those in healthy populations at similar dosages. Thalidomide exhibits a dose-proportional increase in AUC at doses from 50 to 400mg. Because of the low solubility of thalidomide Cmax is less than proportional to dose, and tmax is prolonged with increasing dose. Age, sex and smoking have no effect on the pharmacokinetics of thalidomide, and the effect of food is minimal. Thalidomide does not alter the pharmacokinetics of oral contraceptives, and is also unlikely to interact with warfarin and grapefruit juice. Since thalidomide is mainly hydrolysed and passively excreted, its pharmacokonetics are not expected to change in patients with impaired liver...

The determination of a potential impurity in Thalidomide drug substance and product by HPLC with indirect UV detection.

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.