Development and validation of simultaneous assay of simvastatin, beta-hydroxy simvastatin as metabolite in human plasma using liquid chromatography-tandem mass spectrometry

Original Research

Abstract

Introduction: Several generic products containing simvastatin are circulating on the Vietnamese market at a more inexpensive price than that of a brand-name one. These formulations, however, have not been assessed for in vivo bioequivalence to the reference product. After oral administration, simvastatin (SIM) is extensively converted into an active metabolite, beta-hydroxy simvastatin acid (SIM-A) and a very low concentration of simvastatin can be found in plasma. Therefore, a method for quantification of simvastatin and its metabolite needs to be developed with a high specificity and sensitivity to detect these analytes in human plasma at such low concentrations. Our purpose was to develop a reliable LC-MS/MS (liquid chromatography-tandem mass spectrometry) method for simultaneous determination of simvastatin and metabolite of simvastatin, beta-hydroxy simvastatin acid, in human plasma and to apply this method to evaluate the bioequivalence of a test product in comparison with the reference product.

Methods: Mass spectrometry, internal standard (IS), and chromatographic conditions were investigated to find out the suitable IS and conditions. Human plasma samples were treated by liquid-liquid extraction (LLE). The assay was validated in compliance with US-FDA (United States-Food and Drug Administration), and EMA (European Medicines Agency) guidelines.

Results: LC-MS/MS with electrospray ionization interface in positive (for SIM and lovastatin as IS) and negative (for SIM-A) ionization mode performed under the multiple reaction monitoring mode was used for detection of the analytes. The transition of m/z is 436.00 → 285.15, 435.10 → 319.15, and 404.95 → 199.10 for SIM, SIM-A, and IS, respectively. Tert-buthyl methyl ether was used for extraction of analytes from human plasma by a simple LLE followed by addition of an ammonium acetate buffer. The developed method was fully validated with acceptable selectivity, linearity and linear range, matrix effect, lower limit of quantitation (LLOQ), carryover, dilution integrity, and intra- and inter-day accuracy and precision, free-thaw stability.

Conclusions: The method can be applied for quantification of these compounds in human plasma for in vivo bioavailability and bioequivalence studies. 

Graphical abstract

Validation of a simple HPLC method to quantify methotrexate concentrations in human plasma

Original Research

Abstract

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.
 

Graphical abstract

Validation of a simple HPLC method to quantify mycophenolic acid concentrations in human plasma

Original Research

Abstract

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.

Graphical abstract

Prevalence and risk factors of Ureplasma urealyticum and Mycoplasma genitalium among women with secondary infertility in Vietnam – A cross-sectional study

Clinical Article

Abstract

Introduction: Ureaplasma urealyticum and Mycoplasma genitalium are infectious pathogens resulting in non-gonococcal urethritis and complications such as pelvic inflammatory disease (PID) and infertility. This study aimed to determine the prevalence of U. urealyticum and M. genitalium in women with secondary infertility and the related factors to these infections.

Methods: This cross-sectional descriptive study was carried out from July 2017 to June 2018. Cervical specimens were collected from women with secondary infertility at the Center for Reproductive Endocrinology and Infertility, Hue University Hospital, Vietnam. PCR was applied for detection of U. urealyticum and M. genitalium. Tubal patency was assessed by hysterosalpingography.

Results: Prevalence of U. urealyticum and M. genitalium were 37.9% and 2.1%, respectively. The association was not statistically significant among infection and the following factors like age, educational level, occupation, history of miscarriage, history of genital infection and abdominal surgery, or infertility duration (p > 0.05). There was a statistically significant correlation between U. urealyticum infection and tubal damage according to hysterosalpingography (p < 0.05).

Conclusion: In the case of women with secondary infertility, genital infection with M. genitalium was rare, whereas that with U. urealyticum infection was high and appeared to be associated with tubal damage.

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