Validation of a Liquid Chromatography/Tandem Mass Spectrometry Assay for the Quantification of Plasma Dihydroartemisinin

Background: Insufficient plasma level of dihydroartemisinin (DHA) can select resistance and will further hinder malaria elimination program. We investigated clinical applicability of a validated liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay to quantify plasma concentration of DHA in healthy subjects from a single oral administration of fixed dose combination of Dihydroartemisinin-Piperaquine.Materials and Methods: Micro-elution solid-phase extraction in a 96-well plate format was used to prepare the samples. DHA separation happened in Acquity UPLCTM BEH C18 column (50 × 2.1 mm, 1.7 µm). Mobile phase was a mixture of acetonitrile-ammonium acetate 10 mM pH 3.5 (50:50, v/v) at 0.3 mL/minute flow rate. Waters Acquity UPLC™ H-Class system coupled with triple quadruple mass spectrometry in positive electrospray ionization mode was used for detection. The internal standard was a stable isotope labelled DHA.Results: Calibration curve was linear with a correlation coefficient >0.995 over a concentration range of 1–1,000 ng/mL. Bias and variation for accuracy and precision were in the range of 15% (20% at the lower limit of quantification). Using 5 µL sample, lower limit of quantification was 1 ng. Matrix effect was less than 15%. The method was successfully applied to investigate the pharmacokinetics of DHA from five healthy subjects, although carry over and the role of anticoagulants were not tested.Conclusion: The LC-MS/MS assay for the quantification of plasma DHA was validated for selectivity, linearity, lower limit of quantitation, accuracy, precision, matrix effect and stability. Although clinical applicability was demonstrated, this method was to be improved to address the not-tested validation parameters.Keywords: dihydroartemisinin, liquid chromatography/tandem mass spectrometry assay (LC-MS/MS), malaria, Indonesia

Pharmacokinetics of cannabichromene in a medical cannabis product also containing cannabidiol and Δ9-tetrahydrocannabinol: a pilot study

Purpose Cannabichromene (CBC) is a phytocannabinoid commonly found in cannabis, yet its acute post-dose pharmacokinetics (PK) have not been examined in humans. This is a secondary data analysis from a trial investigating Spectrum Yellow oil, an oral cannabis product used for medical purposes that contained 20 mg cannabidiol (CBD), 0.9 mg Δ9-tetrahydrocannabinol (THC), and 1.1 mg CBC, per 1 mL of oil. Methods Participants (N = 43) were randomized to one of 5 groups: 120 mg CBD, 5.4 mg THC, and 6.6 mg CBC daily; 240 mg CBD, 10.8 mg THC, and 13.2 mg CBC daily; 360 mg CBD, 16.2 mg THC, and 19.8 mg CBC daily; 480 mg CBD, 21.6 mg THC, and 26.4 mg CBC daily; or placebo. Study medication was administered every 12 h for 7 days. Plasma CBC concentrations were analyzed by a validated two-dimensional high-performance liquid chromatography–tandem mass spectrometry assay. Results After a single dose and after the final dose, the Cmax of CBC increased by 1.3–1.8-fold for each twofold increase in dose; the tmax range was 1.6–4.3 h. Based on the ratio of administered CBD, THC, and CBC to the plasma concentration, the dose of CBD was 18 times higher than the dose of CBC, yet the AUC0–t of CBD was only 6.6–9.8-fold higher than the AUC0–t of CBC; the dose of THC was similar to the dose of CBC, yet THC was quantifiable in fewer plasma samples than was CBC. Conclusions CBC may have preferential absorption over CBD and THC when administered together. Trial Registration: Australian New Zealand Clinical Trials Registry #ACTRN12619001450101, registered 18 October 2019.

Metabolic Profile in Neonatal Pig Hearts

We evaluated the metabolic profile in pig hearts at postnatal day 1, 3, 7, and 28 (P1, P3, P7, and P28, respectively) using a targeted liquid chromatography tandem mass spectrometry assay. Our data showed that there is a clear separation of the detected metabolites in P1 vs. P28 hearts. Active anabolisms of nucleotide and proteins were observed in P1 hearts when cardiomyocytes retain high cell cycle activity. However, the active posttranslational protein modification, metabolic switch from glucose to fatty acids, and the reduced ratio of collagen to total protein were observed in P28 hearts when cardiomyocytes withdraw from cell cycle.

Newborn Screening for Krabbe Disease—Illinois Experience: Role of Psychosine in Diagnosis of the Disease

Population-based newborn screening for Krabbe disease was initiated by measurement of galactocerebrosidase (GALC) activity in the state of Illinois in December 2017. Due to the poor specificity of GALC for the diagnosis of Krabbe disease, second-tier testing services were provided to reduce the false positive rates for disease monitoring. Using ultra-pressure liquid chromatography coupled to mass spectrometry assay, a total of 497,147 newborns were screened. In total, 288 infants’ specimens (0.06%) having reduced GALC activity were sent out for second-tier testing to a reference laboratory. All newborns’ reduced GALC specimens were tested for psychosine levels, the presence of a 30-kb deletion and GALC sequencing. The results showed that two infants had elevated psychosine levels (10 and 35 nM) and were referred immediately for evaluation and treatment for Infantile Krabbe disease, and six infants had intermediate PSY levels (≥2 to 5 nM) and are under observation as suspected candidates for late-onset Krabbe disease. In addition, 178 infants had pseudodeficiency alleles, all having psychosine levels < 2.0 nM. Our data show that a high percentage of reduced GALC activity (62%) was due to the presence of pseudodeficiency alleles in the GALC gene. In conclusion, incorporation of psychosine measurements can identify infants with infantile Krabbe disease and probable late-onset Krabbe infants. Furthermore, Krabbe disease screening can be achieved at public health laboratories, and infants with infantile Krabbe disease can be diagnosed in timely manner for better outcome.