生物分析および生物医学ジャーナル

Auto-reactive cells which escape from natural apoptosis represent a continuous threat of potential autoimmune response. Abnormal apoptosis can play a role in negative selection of B and T lymphocytes that escaped the selfreactive nature, and so, apoptosis could represent an additional source of auto-antibody. Increased activity of T cells(CD3 +, CD4 +, or Th1 helper)) will, at a high serum level, cause a high expression of various types of inflammatory interleukins: IL1-β, IL2. The most important regulatory mechanisms of apoptosis in T and B cells are: death receptor cells, CD 95(Fas), TNF tumor necrosis, caspases, family Bcl-2 proto-oncogenes, Bax gene, p53 tumor suppressor gene, and NF-κB nuclear factor of transcription and micro RNAs (miRNAs).The “decision” to undergo programmed cell death is made only in the presence of extrinsic or intrinsic apoptotic messengers. Extrinsic inductors are ligands – cytokines – that bind to death receptors (DRs) found on the cells’ surface, while intrinsic inductors come from the mitochondria or from the nucleus cells.

A simple and reproducible high-performance liquid chromatographic method was developed for simultaneous determination of sulfamethoxazole (SMX) and trimethoprim (TMP), which are also known as cotrimoxazole, in tablet and human plasma in vitro . The Concentration of TMP and SMX used for this study were 1:5 according to their combination as cotrimoxazole. Chromatography was performed on a C 18 column (250 mm × 4.6 mm, 5 μm) under isocratic elution with acetonitrile-water-triethylamine (20:80:0.1 v/v), pH 5.9 ± 0.1 arranged by 0.2 N NaOH or 1% acetic acid. Detection was made at 240 nm and analyses were run at a flow-rate of 1.0 ml/min at a room temperature. Sulfadimidine was used as internal standard. In tablet validation, the calibration curve was linear by r values 0.9994 and 0.9996, precision by coefficient of variation (CV) were 0.85% and 0.98% also accurate by % recovery for 3 concentrations were 98% - 102% for TMP and SMX, respectively. Plasma extraction was done by deproteination with acetonitrile, mix with vortex for 40s, then centrifuge it on 12500 rpm for 15 minutes. In plasma validation, the recovery was 94.95% and 86.87% for TMP and SMX, respectively. The lower limit of quantification (LLOQ) in plasma was 150 ng/mL and 750 ng/mL for TMP and SMX, respectively. The method also fulfill the criteria for accuracy and precision intra and inter day by % diff values not exceed ± 20% for LLOQ and ± 15% for concentrations except LLOQ. On the stability study, cotrimoxazole in plasma is pronounced to be stable for 30 days.

The use of blood spot collection cards was investigated as a means of obtaining small volume samples for the analysis of therapeutic drugs for the purpose of neonatal pharmacokinetic studies. We describe the development of two micro-analytical methods for the determination of captopril extracted from dried blood spots (DBS). Firstly a liquid chromatography ion-trap mass spectrometry method with selected ion monitoring (LC-MS(SIM)) of target ion at m/z 218.0 was developed to determine captopril levels in 8mm discs punched from each DBS. This was compared in terms of specificity and sensitivity to a simple accurate mass liquid chromatography high resolution TOF mass spectrometry (LC-HRMS) method in which MS detection was carried out in electrospray positive ion mode for target ions at m/z 218.0845 for captopril and 377.2084 for the IS. Dithiothreitol was used both to pre-treat the sampling cards and as part of the extraction medium in order to stabilise the DBS extracted captopril. Drug extraction efficiency from spiked blood spots was demonstrated to be 90 ± 10% and the drug was stable in DBS for at least 12 weeks. Validation of both micro-analytical methods showed good precision and accuracy and the LC-HRMS method was linear within the tested calibration range 10-400 ng/ml for captopril and had improved sensitivity and specificity compared to the LC-MS(SIM) method. This method was applied to blood spots on sampling card from a neonate patient previously administered 1 mg/kg captopril orally. The amount of captopril in the DBS was 88 ng/ml. Requiring only a micro volume (30 μl) blood sample for analysis, the developed DBS based micro-analytical method has the potential to facilitate pharmacokinetic studies of captopril in children.