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A new high-performance liquid chromatographic method for determination of warfarin enantiomers.
Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Chemistry.
Linköping University, Department of Medicine and Health Sciences, Internal Medicine . Linköping University, Faculty of Health Sciences.ORCID iD: 0000-0003-1068-6168
Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
2005 (English)In: Journal of Chromatography B, ISSN 1570-0232, Vol. 826, no 1-2, 75-80 p.Article in journal (Refereed) Published
Abstract [en]

Warfarin is the most common agent used for control and prevention of venous as well as arterial thromboembolism. Although warfarin is administered as a racemic mixture of two stereoisomers (S and R), the S-form is mainly responsible for the anticoagulant effect. The anticoagulant effect of the drug is monitored by analysis of prothrombin complex (International Normalised Ratio,INR). In some cases, however, the measurements of plasma warfarin concentration are needed. Here, we present a new, rapid, sensitive and cost-effective HPLC-method for the determination of warfarin enantiomers in plasma. The chromatographic system consisted of Waters 616 gradient pump, Waters 996 photo diode array detector, Gilson 230 autoinjector and Pirkle (R,R) Whelk-O1 column (25 cm × 4.6 mm I.D., 5 μm). An isocratic mobile phase of methanol/acetonitrile/water (50/10/40, v/v) with 0.1% glacial acetic acid was used. The follow rate was 1 mL/min. Data analysis was carried out with Waters Millennium32. The absorbance at 305 nm was measured with a total run-time of 15 min. Method linearity was studied by establishing regression data containing eight points over the range 0.08–10 μg/mL. In this range, warfarin showed to be linear (r2 = 0.9997 for S-warfarin and r2 = 0.9998 for R-warfarin). The limit of detection in plasma was 16 ng/mL for S-warfarin and 18 ng/mL for R-warfarin. Limit of quatitation was defined as 10 × LOD. The extraction recovery was approximately 80%. Also the relation between INR and warfarin concentration was investigated. As expected, there was a low correlation between these two variables (r = 0.23, y = 0.3044x + 0.9712). This method offers a rapid and cost-effective determination of warfarin enantiomers in human plasma.

Place, publisher, year, edition, pages
2005. Vol. 826, no 1-2, 75-80 p.
Keyword [en]
S- and R-warfarin; Oxybenzone; INR
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-14487DOI: 10.1016/j.jchromb.2005.08.011OAI: oai:DiVA.org:liu-14487DiVA: diva2:23582
Available from: 2007-05-21 Created: 2007-05-21 Last updated: 2013-09-03
In thesis
1. Studies on warfarin treatment with emphasis on inter-individual variations and drug monitoring
Open this publication in new window or tab >>Studies on warfarin treatment with emphasis on inter-individual variations and drug monitoring
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [sv]

Waran används sedan 60 år som blodförtunnande läkemedel för att förebygga eller förhindra progress av blodproppssjukdom. I Sverige behandlas årligen cirka 1 % av befolkningen med waran. I Östergötland uppskattas antal waranpatienter till cirka 3000. Waran hämmar enzymet VKORC1 som ansvarar för vitamin K omsättningen i kroppen. Vitamin K behövs som kofaktor för flera koagulationsfaktorer.

Behandling med waran är förenad med en svår balansgång och kräver en noggrann dosering. Stora skillnader i dosbehov mellan olika individer, beroende på ärftliga och miljöfaktorer, gör waran till ett svårhanterligt läkemedel. För låg dos medför otillräcklig effekt och därmed risk för minskat skydd mot blodproppssjukdom. För hög dos leder till allvarliga blödningskomplikationer. Uppskattningsvis 1 – 3 % livshotande blödningsfall registreras årligen efter waranbehandling. Därför måste behandlingen kontrolleras noga med analys av protrombinkomplex (PK) och dosjusteringar göras med ledning av resultaten. Två olika metoder finns att använda för mätning av PK. I Norden och i Japan används Owrens metod (utvecklat i Norge under 40- och 50-talet av Paul Owren). I de flesta andra länder används Quickmetoden (utvecklat i USA under 30-talet av Armand Quick). Den senare metoden är förenad med stora variationer mellan olika analyslaboratorier. I Norden, däremot, där Owrens metod används finns det ofta bra överensstämmelse mellan olika laboratorier i PK-resultat. Beroende på vilken PK-metod som används, kan samma patient få olika warandoser vilket ökar risker för under- eller överbehandling.

Vi har i samarbete med flera sjukhus och antikoagulationsmottagningar (AK-mottagningar) i sydöstra Sverige studerat dels mekanismerna bakom skillnader i warandos mellan olika patienter, och dels tittat varför de olika PK-metoder skiljer sig så mycket som de gör.

I studien har vi identifierat genetiska varianter av enzymet VKORC1. Av de undersökta patienter som gick på waran under längre tid, har vi identifierat en grupp som markant skiljde sig från de övriga. Denna grupp hade warandoser som var betydligt lägre än de övriga. När vi kartlade deras arvsmassa, fann vi att lågdospatienterna hade genvarianten VKORC1*2. Dessutom hade patienter med denna variant svårigheter att få stabila PK-värden. De gjorde också fler besök på AK-mottagningar än andra patienter. Vi har därför konstaterat att en del av de problem som är förenade med waranbehnadlingen kan förklaras av VKORC1*2 varianten. Vetskap om denna variant skulle troligen underlätta behandlingen framför allt under inledningsfasen då patienter med VKORC1*2 riskerar blödningar på grund av överdosering.

Vi har identifierat att provförspädning enligt Owrens metod är nödvändig för harmonisering av PK-resultatet mellan olika länder. Quickmetoden använder inte förspädning av patientprov till skillnad från Owrens metod. När vi modifierade en Quickmetod genom att förspäda prover enligt Owrens metod noterade vi en bra överensstämmelse mellan de två olika metoderna. Däremot var resultatet sämre utan provförspädning. Vi anser att Quickmetoder kan uppnå lika bra resultat som Owrens metod om prover förspäds som i Owrens metod. Det skulle gynna patienter som reser mellan olika regioner eller länder och leda till en bättre övervakning av waranbehandling internationellt.

I studien har dessutom en metod för mätning av waran i blodet utvecklats. Metoden som är den enda i sitt slag i Norden ger möjlighet att studera hur läkemedlet beter sig i kroppen. Vi har med denna metod kunnat upptäcka patienter som har onormala nedbrytningar av waran.

Abstract [en]

Warfarin was introduced more than 60 years ago and is used worldwide for the prophylaxis of arterial and venous thromboembolism in primary and secondary prevention. The drug is orally administered as a racemic mixture of (R)- and (S)-enantiomers. The (S)-form is mainly responsible for the anticoagulant effect and is metabolised by CYP2C9 enzyme in the liver microsomes. Warfarin exerts its pharmacological action by inhibiting the key enzyme (VKORC1) that regenerates vitamin K from an oxidised state to a reduced form. The latter is a cofactor for the post-translational modification of a number of proteins including coagulation factors II, VII, IX and X. The vitamin K-dependent modification provides these factors with the calcium-binding ability they require for the interaction with cell membranes of their target cells such as platelets.

Warfarin is monitored by measuring prothrombin time (PT) expressed as INR. Two main methods exist for PT analysis. The Owren method is used mainly in the Nordic and Baltic countries, in Japan, whereas the Quick method is employed in most other countries. Warfarin management is associated with some complications. Unlike many other drugs the dose for a given patient cannot be estimated beforehand, dose-response relationship is not predictable, and the prevention of thrombosis must be balanced against the risk of bleeding. Furthermore, the different PT methods used to monitor the drug are sometimes not in agreement and show significant discrepancies in results.

In an attempt to clarify the mechanisms influencing the inter-individual variations in warfarin therapy and to detect the factors that contribute to differences between PT methods, studies were conducted in collaboration with hospitals and anticoagulation clinics in the south-eastern region of Sweden. First, a stereo-specific HPLC method for measurement of warfarin enantiomers was developed and validated. With this method, the levels of plasma warfarin following its oral administration can be studied and evaluated. Abnormal clearance in some patients can be detected, and patient compliance can be verified. Furthermore, differing ratios of (S)- and (R)-isomers can be identified.

The impact of common VKORC1 polymorphisms on warfarin therapy was investigated. This study has shown that the VKORC1*2 haplotype is an important genetic determinant for warfarin dosage and is associated with difficulties in attaining and retaining therapeutic PT-INR. Further, significant differences in warfarin S/R-ratio was detected between patients with VKORC1*2 and VKORC1*3 or VKORC1*4 variants. This difference was not coupled with CYP2C9 genotype.

The effects of predilution of patient plasma samples, sources of thromboplastin and deficient plasma on between PT methods agreement were studied. This study has revealed that sample predilution according to the Owren method is to be preferred for the harmonisation of PT results. Undiluted samples, in contrast, according to the Quick method have shown reduced correlation between two different thromboplastin reagents. Sources of thromboplastin and deficient plasma were only of minor importance.

Place, publisher, year, edition, pages
Institutionen för biomedicin och kirurgi, 2007. 68 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1000
Keyword
Warfarin, VKORC1, Haplotype, Prothrombin time, INR
National Category
Other Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-8874 (URN)978-91-85715-45-9 (ISBN)
Public defence
2007-06-01, Berzeliussalen, Campus HU, Universitetssjukhuset, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2007-05-21 Created: 2007-05-21 Last updated: 2009-08-22

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