Identification, synthesis and quantification of process-related impurities in auraptene.

Impurities in chemically synthesized auraptene, an active pharmaceutical ingredient (API), were detected by a gradient reverse-phase high-performance liquid chromatography (RP-HPLC) method. Molecular weights and major product ions of these chemical compounds were determined by liquid chromatography/Triple Quadrupole (LC-MS/MS) analysis. Structural assignments were presumed as umbelliferone (Imp-I), (E)-6,7-dihydroxy-3,7-dimethyl-2-octene-umbelliferone (Imp-II), (E)-6,7-epoxy-3,7-dimethyl-2-octene-umbelliferone (Imp-III) and 4-methylauraptene (Imp-IV). The impurities were authentically synthesized, confirmed by nuclear magnetic resonance spectroscopy (NMR) and infrared spectroscopy (IR), and subsequently used as reference samples in routing HPLC system suitability testing for method specificity and detectability. Method specificity was further verified by forced degradation studies. The developed method was validated for characterization of impurities in synthesized auraptene according to the guidelines of the International Conference on Harmonization (ICH) in our laboratory.

Toxicology and risk assessment of coumarin: focus on human data.

Coumarin is a secondary phytochemical with hepatotoxic and carcinogenic properties. For the carcinogenic effect, a genotoxic mechanism was considered possible, but was discounted by the European Food Safety Authority in 2004 based on new evidence. This allowed the derivation of a tolerable daily intake (TDI) for the first time, and a value of 0.1 mg/kg body weight was arrived at based on animal hepatotoxicity data. However, clinical data on hepatotoxicity from patients treated with coumarin as medicinal drug is also available. This data revealed a subgroup of the human population being more susceptible for the hepatotoxic effect than the animal species investigated. The cause of the high susceptibility is currently unknown; possible mechanisms are discussed. Using the human data, a TDI of 0.1 mg/kg body weight was derived, confirming that of the European Food Safety Authority. Nutritional exposure may be considerably, and is mainly due to use of cassia cinnamon, which is a popular spice especially, used for cookies and sweet dishes. To estimate exposure to coumarin during the Christmas season in Germany, a telephone survey was performed with more than 1000 randomly selected persons. Heavy consumers of cassia cinnamon may reach a daily coumarin intake corresponding to the TDI.

Coumarins permeability in Caco-2 cell model.

OBJECTIVES: The presence of coumarins in human diet, their multiple pharmacological properties and occurrence in various herbal remedies represent significant reasons to explore their membrane permeability, as a first event contributing to coumarins oral bioavailability. Thus, we evaluated the permeability and cytotoxicity of 18 coumarins, with different substitution patterns involving OH, OCH3 and CH3 groups. METHODS: A modified Caco-2 permeability model was used, in which the permeability test is performed with a robotic workstation and cells are grown on 96-well plates for 7 days. KEY FINDINGS: All studied coumarins were highly permeable, with calculated Papp values that varied within 4.1 x 10(-5) to 2.1 x 10(-4) in apical to basolateral studies and within 1.8 x 10(-5) to 7.0 x 10(-5) in basolateral to apical studies. The efflux ratio remained in all cases below 1. It was demonstrated that the type and position of substituents contributed more to the permeability than the number of substituents. CONCLUSIONS: The results allowed us to predict that these coumarins are well absorbed in the gut lumen and efflux is not limiting the absorption. Five coumarins had an influence on the mitochondrial function of Caco-2 cells (1 < 80%, 4 > 120%), according to the WST-1 cytotoxicity test, but this does not seem to affect the permeability of the compounds.

Separation and determination of warfarin enantiomers in human plasma using a novel polymeric surfactant for micellar electrokinetic chromatography-mass spectrometry.

Warfarin is a widely used oral anticoagulant which is mostly administrated as a racemic mixture containing equal amount of R- and S-enantiomers. The two enantiomers are shown to exhibit significant differences in pharmacokinetics and pharmacodynamics. In this study, a new chiral micellar electrokinetic chromatography-mass spectrometry (MEKC-MS) method has been developed using a polymeric chiral surfactant, polysodium N-undecenoyl-L,L-leucyl-valinate (poly-L,L-SULV), as a pseudostationary phase for the chiral separation of (+/-)-warfarin (WAR) and (+/-)-coumachlor (COU, internal standard). Under optimum MEKC-MS conditions, the enantio-separation of both (+/-)-WAR and (+/-)-COU was achieved within 23 min. Calibration curves were linear (R=0.995 for (R)-WAR and R=0.989 for (S)-WAR) over the concentration range 0.25-5.0 microg/mL. The MS detection was found to be superior over the commonly used UV detection in terms of selectivity and sensitivity with LOD as low as 0.1 microg/mL in human plasma. The method was successfully applied to determine WAR enantiomeric ratio in patients' plasma undergoing warfarin therapy.

Adherence to a guideline for coumarins in pregnancy.

OBJECTIVE: To asses the adherence in daily clinical practice to a guideline for anticoagulation during pregnancy. METHODS: The Dutch anticoagulation clinics developed a pregnancy guideline for anticoagulant therapy in order to avoid foetal exposure to coumarins between the 6th and 9th week of gestation. Anticoagulation was studied in 282 prospectively-registered pregnant women, who were treated by 26 different anticoagulation clinics. RESULTS: The guideline was adhered to in 93% of treated women. Conforming to the guideline, the majority of patients commenced anticoagulation with heparin in the first trimester (n = 81) or started treatment from the second trimester onwards (n = 168). At the time of conception, 31 anticoagulated women were on coumarin treatment. In 13 of these patients (42%), coumarins were withdrawn before the 6th gestational week. In two pregnant women coumarin therapy started unintentionally during the first trimester of gestation. CONCLUSION: The present study shows that the guideline under study is useful in daily clinical practice. A careful instruction of women of child-bearing age who need medication remains important.

Stability studies on lyophilised reference thromboplastins for standardisation of prothrombin-times.

An international framework for anticoagulant control has been developed based on British Comparative Thomboplastin (B.C.T.) and the model provided by the British system for anticoagulant control. An alternative international system has been proposed, based on lymphilised thromboplastins prepared at the National Institute of Biological Standards and Control, London. Since 1969, stability studies on two of the N.I.B.S.& C. reagents, the primary material 67/40 and the secondary thromboplastin 69/223, have been in progress at the National (U.K.) Reference Laboratory for Anticoagulant Reagents and Control, Manchester. Both the proposed N.I.B.S.&C. reference preparations have deteriorated, while two lyophilised reagents prepared at the National (U.K.) Reference Laboratory have revealed no evidence of instability. In national and international standardisation reliance on B.C.T. should continue.