Methotrexate (MTX) is a chemotherapy and immunosuppressive agent widely used to treat cancer, autoimmune diseases in children and adult patients, and ectopic pregnancy. However, MTX is highly toxic to the liver, kidney, and nervous system. This study aimed to quantify the concentration of MTX in human plasma using high-performance liquid chromatography (HPLC). MTX and its internal standard (para aminoacetophenone-PAPA) in plasma samples were extracted simultaneously with methanol. Sample purity was performed using the 1 cc OASIS HLB cartridges. Sample injection volume of 10 µL was analyzed on a Lichrocart Supersil 125-4 column C18 maintained at 40 °C on a Waters 2695 XE equipped with a PDA detector set at 303 nm. The mobile phase contained phosphate buffer (pH 6.0) and methanol at a ratio of 80:20 (v/v) and was maintained at a flow rate of 1 ml/min. The results showed that the total time of chromatographic analysis was 15 min. MTX and PAAP were found in the chromatograms at retention times of 2.3 and 5.2 min, respectively. The linear range of the MTX from 0.5 to 25 µg/mL. Intra-day and inter-day imprecision for MTX ranged from 3.42 to 8.128%. LLOQ of MTX was 0.5 µg/mL and the extraction effects were above 77%. In conclusion, we developed and validated a simple HPLC method to determine the MTX concentrations in human plasma.
 

Introduction: Mycophenolic acid (MPA) is an active metabolite of mycophenolate mofetil and mycophenolate sodium which are widely prescribed to prevent organ rejection after solid organ transplantations. However, MPA induced many side effects on gastrointestinal tract and haematological system.

Objectives: The purpose of this study is to establish a high-performance liquid chromatography (HPLC) method to determine the MPA concentration in plasma in order to optimize the treatment efficacy of MPA or apply to bioequivalence studies. MPA and visnadine (as an internal standard) were extracted from plasma samples with methanol by solid phase extraction using Osis HLB 1cc cartridge. 10 µL of sample extract was injected onto LiChroCART®125-4 (C18 reversed-phase column) at 43 ^°C on a Waters 2695 XE system. The signals were detected by PDA detector (photodiodes array) at 254 nm. The mobile phase was a mixture of acetonitrile and phosphate buffer (pH 3) with a flow rate of 1 mL/min. The validation criteria included: selectivity, linearity, accuracy, precision, recovery, lower limit of quantification.

Results: Total chromatographic runtime was 15 min. MPA and visnadine were found at 6.45 and 10.79 min, respectively. MPA concentrations were in the linear range from 0.25 to 50 µg/mL. The coefficient of variation (CV) of mean intra-day and inter-day precision levels for MPA was less than 7.5%. The lower limit of quantification was 0.25 µg/mL. No interference was found in the assay.

Conclusion: A simple and reliable HPLC method was developed to quantify the MPA concentration in plasma.

Many plant parts have been widely used in the treatment of immune diseases in Vietnam, yet just few of them are known about their mechanism of action. The goal of this study was to investigate the in vitro inhibitory effects of in vitro inhibitory effect of crude ethanol extracts and fractions of thirteen medicinal plants on proliferation of human peripheral blood mononuclear cells (PBMCs), interleukin-2 secretion and antioxidant activity. PBMCs were extracted from the whole blood of healthy volunteers. The effects of thirteen crude extracts in ethanol and twenty four fractionated extracts in chloroform, ethyl acetate and water on in vitro proliferation of PBMCs were evaluated using MTT test. Interleukin - 2 (IL - 2) concentrations secreted by PBMC were determined by ELISA method. The antioxidant capacity of ethanol extracts and fractionated extracts were assessed using the DPPH method. The results showed that out of thirteen crude ethanol extracts, six extracts inhibited PBMC proliferation and two extracts stimulated PBMC proliferation and five extracts had no effect on PBMC proliferation. The inhibitory extracts reduced the amount of IL-2, while the stimulant extracts while the stimulant extracts had no effect on IL-2 secretion compared to the control. The chloroform extract of Wedelia chinesis showed the strongest inhibitory activity with an IC₅₀ concentration 16.1 µg/ml. The chloroform extract of Piper betle showed the strongest DPPH capture capacity with DPPH₅₀ 1.94 µg/ml and 2.1 times stronger than vitamin C. In conclusion, the chloroform extract of Wedelia chinensis may be considered for the treatment of autoimmune diseases.