The antimalarial lumefantrine was first synthesised and registered in China and is now commercially available as a coformulated product together with artemether (Coartem®/Riamet®). This combination is well tolerated and has proven highly efficacious for treatment of uncomplicated falciparum malaria. Lumefantrine is highly lipophilic with an extensive protein binding (99.9%). The day 7 plasma lumefantrine level has been shown to be an important determinant of treatment efficacy. To date no method has been published for the determination of lumefantrine after capillary sampling onto filter paper for field use. The aim of this work was to develop a method with adequate sensitivity for quantification of lumefantrine in capillary blood sampled onto filter paper. The method has been validated according to the current FDA guideline for bioanalytical method validation. Method: Whatman 31 ET Chr filter paper was pre-treated with an organic acid before sampling capillary blood to enable a high recovery of lumefantrine. Lumefantrine was extracted from the filter paper, then further purified using solid phase extraction and finally quantified with HPLC. Results: The between day variation is below 10 % over the range 0.4 to 25 µmol/l. The lower limit of quantification is 0.25 µmol/l in 100 µl capillary blood. No decrease in Lumefantrine concentration in dried blood spot is seen after 3 months at 37o C. The field sampling for lumefantrine assay with pre-treated Whatman 31 ET Chr has been tested in Tanzania with good results. Discussion: The field sampling for lumefantrine concentration assay with pre-treated Whatman 31 ET Chr has been evaluated and proven to be a valid method for field studies. The day 7 level after treatment can lumefantrine be accurately estimated in capillary blood to follow up compliance and efficacy. Validation data will be presented.
A bioanalytical method for the determination of lumefantrine in 100 µl blood applied onto sampling paper, by solid-phase extraction and liquid chromatography, has been developed and validated. Whatman 31 ET Chr sampling paper was pre-treated with 0.75 M tartaric acid before sampling capillary blood to enable a high recovery of lumefantrine. Lumefantrine was extracted from the sampling paper, then further purified using solid-phase extraction and finally quantified with HPLC. The between-day variation was below 10% over the range 0.4–25 µM. The lower limit of quantification was 0.25 µM in 100 µl capillary blood. No decrease in lumefantrine concentration in dried blood spot is seen after 4 months storage at 22 °C. The method was also evaluated in field samples from patients in Tanzania after treatment with lumefantrine/artemether. Lumefantrine could be estimated accurately enough to assess bioavailability and treatment compliance on day 7 (i.e. 4 days after the last dose) after a standard regimen with the lumefantrine/artemether combination.
Background: More parasites are becoming resistant to antimalarial drugs, and in many areas a change in first-line drug treatment is necessary. The aim of the developed assay is to help determine drug use in these areas and also to be a complement to interviewing patients, which will increase reliability of surveys.
Results: This assay detects quinine, mefloquine, sulfadoxine, pyrimethamine, lumefantrine, chloroquine and its metabolite desethylchloroquine in a 100-mu l dried blood spot. Most of the drugs also have long half-lives that make them detectable at least 7 days after administration. The drugs are extracted from the dried blood spot with sequential extraction (due to the big differences in physicochemical properties), solid-phase extraction is used as sample clean-up and separation is performed with gradient-LC with MS ion-trap detection.
Conclusion: Detection limits (S/N > 5:1) at 50 ng/ml or better were achieved for all drugs except lumefantrine (200 ng/ml), and thus can be used to determine patient compliance. A major advantage of using the ion-trap MS it that it will be possible to go back into the data and look for other drugs as needed.
A bioanalytical method for indirect determination of eflornithine enantiomers in 75?µL human plasma has been developed and validated. l- and d-eflornithine were derivatized with o-phthalaldehyde and N-acetyl-L-cysteine to generate diastereomers which were separated on two serially connected Chromolith Performance columns (RP-18e 100 × 4.6?mm i.d.) by a isocratic flow followed by a gradient flow for elution of endogenous compounds. The diastereomers were detected with UV (340?nm). The between-day precisions for L- and D-eflornithine in plasma were 8.4 and 2.3% at 3?µm, 4.0 and 5.1% at 400?µm, and 2.0 and 3.7% at 1000?µm. The lower limit of quantification was determined to be 1.5?µm, at which precision was 14.9 and 9.9% for L- and D-eflornithine, respectively
Projektet är ett samarbete mellan Forskare vid Högskolan Dalarna och en kommunal skola i Dalarna. På aktuell skola fanns nio klasser och ca 20 lärare varav alla mer eller mindre deltog i projektet. Syftet med projektet var att skapa kunskap om, samt utveckla, undervisning och processer som kan synliggöra kunskaper och färdigheter inom naturorienterande ämnen (NO) i grundskolans tidigare årskurser, förskoleklass till årskurs 6 (F-6). Studiens frågeställning var; Hur kan undervisning inom ämnesområdet NO utvecklas och hur kan verktyg som lärare använder för att stödja elever i deras arbete utvecklas och synliggöras?
Som metod valdes aktionsforskning och projektet valde att arbeta med en fokusgrupp bestående av tre lärare från Högskolan Dalarna samt av lärare från aktuell skola. Genom kollektiva diskussioner identifieras områden i lärarnas verksamhet som kunde utvecklas, diskussionerna ledde fram till en aktion (medveten förändring) och lärarna genomförde därefter undervisning som inbegrep de nya, medvetna förändringarna. Utfallet av dessa förändringar analyserades sedan gemensamt inom fokusgruppen. Inom projektets ram videoinspelades de NO-lektioner som var i fokus för utveckling. Vetenskapsrådets (2002; 2010) forskningsetiska riktlinjer har beaktats under forskningsprocessen. Alla medverkande i projektet informerades om projektets syfte och genomförande både muntligt och skriftligt. Totalt genomfördes tre aktionsforskningscykler från hösten 2013 till hösten 2015. Den första aktionsforskningscykeln genomfördes med utgångspunkt i ämnet kemi och med fokus på att inkludera praktiska moment som förtydligade och konkretiserade innehållet i undervisningen. Vid utvärderingen av undervisningen identifierades flera problemområden. Två av dessa områden valdes för aktionscykel två, hur experiment och laborationer kan knytas till teori och betydelsen av att lärare använder både vardagliga och naturvetenskapliga begrepp. Utvärderingen av aktionscykel två resulterade i en tredje aktionsforskningscykel med fokus på systematiska undersökningar. Varje aktionsforskningscykel resulterade i ett antal verktyg som fokusgruppen ansåg som betydelsefullt och som behöver beaktas i undervisningen för elevers lärande i NO. De verktyg som framkom kan beskrivas som att de handlade om att planera och förklara, lyssna in och involvera eleverna samt den skriftliga kommunikationens funktion.
A bioanalytical method for determination of lamivudine (3TC), zidovudine (AZT), and nevirapine (NVP) in 100 μL capillary blood applied onto sampling paper has been developed and validated. The antiretroviral drugs (ARV) were analyzed by reversed phase gradient liquid chromatography with UV detection. Separation was performed on a Zorbax SB C8 (250 × 4.6 mm) column with a twostep gradient: (i) methanol.0.05 mol/L acetic acid-sodium acetate buffer (pH 3.95, 15:85 v/v) and (ii) methanol.0.05 mol/L acetic acid-sodium acetate buffer (pH 3.95, 50:50 v/v) with a flow rate of 1.0 mL/min. UV detection was performed at 260 nm. Total assay precisions were 6.3, 4.7, and 4.9% for 3TC at 0.34, 0.69, and 3.9 μg/mL, and 5.1, 5.5, and 3.2% for AZT at 0.40, 0.80, and 4.5 μg/mL. For NVP, total assay precisions were 5.2, 8.3, and 3.5% at 2.6, 4.5, and 8.8 μg/mL. Lower limit of quantifications (LLOQ) were 0.11 and 0.13 μg/mL for 3TC and AZT where the precisions were 2.0% for both the analytes. For NVP, LLOQ was 1.3 μg/mL where precision was 2.6%. Concentrations were determined for 10 h for two subjects receiving standard twice daily antiretroviral therapy containing 3TC, AZT, and NVP. Maximum 3TC concentrations were 2.5 and 2.8 μg/mL for subject 1 and 2, respectively. For AZT, maximum concentrations were 1.8 and 1.1 μg/mL while being 15 and 9.6 μg/mL for NVP. Pre-dose trough concentration of NVP was 11 μg/mL for subject 1 and 9.6 μg/mL for subject 2.
Malaria is an infectious decease caused by a one-celled parasite called Plasmodium. The most common types are falciparum and vivax malaria where falciparum is the deadliest form of malaria infection. Malaria is present in almost all tropical and sub-tropical regions around the world and there are at least 300 million cases of malaria in the world with over a million deaths every year. Some of the most frequently used antimalarial drugs are chloroquine, amodiaquine and sulphadoxine-pyrimethamine but drug resistance of the plasmodium falciparum parasite is spreading. The situation is especially serious in Southeast Asia where multi-drug resistant falciparum malaria is widespread. One factor that adds to drug resistance is poor compliance with dosing schedules. There are also increasing problems with counterfeit drugs that contain none or to low amount of antimalarial drug and this will also speed up drug resistance. It has recently become more common to combine different antimalarial drugs, preferably using drugs with different mechanisms of action, in order to increase efficacy and reduce the spread of malarial drug resistance within the malaria parasite populations. The aim of the developed screening method is to determine drug use in areas where a change in treatment policy has taken place since this would be more accurate than interviewing patients. Several of the drugs included in this screening method have relative long half-life e.g.chloroquine, pyrimethamine, mefloquine, and lumefantrine that are detectable at least 7 days after administration. Solid phase extraction is used as sample clean-up of plasma samples and separation is performed with gradient-LC and UV-detection at two different wavelengths. The developed method will be presented together with detection limits of the various drugs.
Background: More parasites are becoming resistant to antimalarial drugs, and in many areas a change in first-line drug treatment is necessary. The aim of the developed assay is to help determine drug use in these areas and also to be a complement to interviewing patients, which will increase reliability of surveys. Results: This assay detects quinine, mefloquine, sulfadoxine, pyrimethamine, lumefantrine, chloroquine and its metabolite desethylchloroquine in a 100-µl dried blood spot. Most of the drugs also have long half-lives that make them detectable at least 7 days after administration. The drugs are extracted from the dried blood spot with sequential extraction (due to the big differences in physicochemical properties), solid-phase extraction is used as sample clean-up and separation is performed with gradient-LC with MS ion-trap detection. Conclusion: Detection limits (S/N > 5:1) at 50 ng/ml or better were achieved for all drugs except lumefantrine (200 ng/ml), and thus can be used to determine patient compliance. A major advantage of using the ion-trap MS it that it will be possible to go back into the data and look for other drugs as needed
A sensitive bioanalytical method for the determination of melatonin in saliva by solid-phase extraction (SPE), high-performance liquid chromatography (HPLC) and fluorescence detection has been developed and validated. Saliva was collected with a Salivette((R)) sampling device (Sarstedt) and a mixed-mode SPE column was used for the extraction of melatonin and internal standard (N-acetyl-6-methoxytryptamine) from the saliva. Chromatographic separation was performed using a HyPurity C18 LC column (150x2.1 mm) with mobile phase acetonitrile-ammonium hydrogen carbonate buffer, 0.015 M, pH 6.8 (23:77, v/v). Excitation and emission wavelengths were set to 285 nm and 345 nm, respectively. The within-day precision for the method at 50 pmol/L was 7.9% and the between-day precision was 10.5%. The limit of quantification was 50 pmol/L.
Background: The growing problem of parasites developing resistance to the traditional antimalarial drugs makes the development of new effective and safe drugs crucial. Tafenoquine is a new promising antimalarial drug for prophylaxis and treatment.
Results: A bioanalytical method for the determination of tafenoquine in 100 mu l of capillary blood applied onto sampling paper and in 100 mu l of plasma has been developed and validated. The Whatman 31 ET Chr paper was treated with 0.6 mol/l tartaric acid to improve the extraction recovery and solid-phase extraction was used for cleanup procedure of the blood samples. Plasma samples were precipitated with methanol. Tafenoquine and internal standard were separated on a Zorbax SB-CN column by reversed-phase LC and detected with fluorescence detection at 262 and 470 nm. The within- and between-day variations were below 10 and 14%, respectively, over the range 50-200 nmol/l for capillary blood on sampling paper and below 6 and 10% for plasma samples. The LLOQ of the method was 50 nmol/l.
Conclusion: The developed method has adequate sensitivity and is highly suitable for clinical studies in dried blood spots and plasma.