High-throughput quantitation method for amodiaquine and desethylamodiaquine in plasma using supported liquid extraction technology.

Amodiaquine is a drug used for treatment of malaria and is often used in combination with artesunate in areas where malaria parasites are still susceptible to amodiaquine. Liquid chromatography tandem-mass spectrometry was used to quantify amodiaquine and its active metabolite, desethylamodiaquine, in plasma samples. A low sample volume of 100 µl, and high-throughput extraction technique using a supported liquid extraction (SLE+) technique on an automated liquid handler platform for faster sample processing are some of the advantages of this method. Separation of amodiaquine from desethylamodiaquine was achieved using a reversed phase Zorbax SB-CN 50 mm × 4.6 mm, I.D. 3.5 µm column with acetonitrile and 20 mM ammonium formate with 1% formic acid pH ~ 2.6 (15-85, v/v) as mobile phase. The absolute recoveries of amodiaquine and desethylamodiaquine were 66% to 76%, and their isotope label internal standard were in the range of 73% to 85%. Validation results of the developed method demonstrated intra-batch and inter-batch precisions within the acceptance criteria range of ± 15.0%. There were no matrix or carry-over effects observed. The lower limit of quantification was 1.08 ng/ml for amodiaquine and 1.41 ng/ml for desethylamodiaquine. The method showed robust and accurate performance with high sensitivity. Thus, the validated method was successfully implemented and applied in the evaluation of a clinical trial where participants received artemether-lumefantrine plus amodiaquine twice daily for three days (amodiaquine dose of 10 mg base/kg/day).

High sensitivity methods to quantify chloroquine and its metabolite in human blood samples using LC-MS/MS.

AIM: Chloroquine is an antimalarial drug used in the treatment of Plasmodium vivax malaria. Three methods to quantify chloroquine and its metabolite in blood matrices were developed and validated. Methodology & results: Different high-throughput extraction techniques were used to recover the drugs from whole blood (50 µl), plasma (100 µl) and dried blood spots (15 µl as punched discs) followed by quantification with LC-MS/MS. The intra- and inter-batch precisions were below 15%, and thus meet regulatory acceptance criteria. CONCLUSION: The developed methods demonstrated satisfactory validation performance with high sensitivity and selectivity. The assays used simple and easy to automate extraction techniques. All methods were reliable with robust performance and demonstrated to be suitable to implement into high-throughput routine analysis of clinical pharmacokinetic samples.

Quantification of the antimalarial drug pyronaridine in whole blood using LC-MS/MS - Increased sensitivity resulting from reduced non-specific binding.

Malaria is one of the most important parasitic diseases of man. The development of drug resistance in malaria parasites is an inevitable consequence of their widespread and often unregulated use. There is an urgent need for new and effective drugs. Pyronaridine is a known antimalarial drug that has received renewed interest as a partner drug in artemisinin-based combination therapy. To study its pharmacokinetic properties, particularly in field settings, it is necessary to develop and validate a robust, highly sensitive and accurate bioanalytical method for drug measurements in biological samples. We have developed a sensitive quantification method that covers a wide range of clinically relevant concentrations (1.5ng/mL to 882ng/mL) using a relatively low volume sample of 100µL of whole blood. Total run time is 5min and precision is within ±15% at all concentration levels. Pyronaridine was extracted on a weak cation exchange solid-phase column (SPE) and separated on a HALO RP amide fused-core column using a gradient mobile phase of acetonitrile-ammonium formate and acetonitrile-methanol. Detection was performed using electrospray ionization and tandem mass spectrometry (positive ion mode with selected reaction monitoring). The developed method is suitable for implementation in high-throughput routine drug analysis, and was used to quantify pyronaridine accurately for up to 42days after a single oral dose in a drug-drug interaction study in healthy volunteers.

Heat stabilization of blood spot samples for determination of metabolically unstable drug compounds.

BACKGROUND: Sample stability is critical for accurate analysis of drug compounds in biosamples. The use of additives to eradicate the enzymatic activity causing loss of these analytes has its limitations. RESULTS: A novel technique for sample stabilization by rapid, high-temperature heating was used. The stability of six commercial drugs in blood and blood spots was investigated under various conditions with or without heat stabilization at 95°C. Oseltamivir, cefotaxime and ribavirin were successfully stabilized by heating whereas significant losses were seen in unheated samples. Amodiaquine was stable with and without heating. Artemether and dihydroartemisinin were found to be very heat sensitive and began to decompose even at 60°C. CONCLUSION: Heat stabilization is a viable technique to maintain analytes in blood spot samples, without the use of chemical additives, by stopping the enzymatic activity that causes sample degradation.