| Amino acid, which is the basic unit of proteins, is one of the important compounds to human being. The body relies upon a complex and well-regulated system to maintain blood plasma free amino acids in a steady state concentration. This steady state reflects a balance between the processes of utilization or catabolism and ingestion or biosynthesis. Pathophysiology affecting protein and amino acid metabolism are often accompanied by changes in this steady state, which often result in diseases. In addition, amino acids preparations have been widely used to treat patients with specific diseases or for nutritional support. Thus, changes in plasma amino acids concentration are critically important for diseases diagnosis and can be used to monitor the effects of pharmacotherapies and thus provide insight into the possible mechanism of action of these treatments.It has always been an important theme in the frontier of analytical chemistry to develop widely-suited analysis method for amino acids with fast, high sensitivity, good separation as well as good reproducibility.The purpose of this study was to establish a rapid hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS) method for the determination of plasma free amino acids, which could be applied in non-clinical pharmacokinetic study of amino acids preparations. The present study preliminarily evaluated the pharmacokinetic behavior of Orn administered a single dose of L-Ornithine·α-Ketoglutarate (α-KGO) by po or iv in mice.Five representative amino acids, including Glu (acidic amino acid), Val (neutral amino acid, branched-chain amino acid), Orn (basic amino acid, straight-chain amino acid, non-protein amino acid), Phe (aromatic amino acid, neutral amino acid) and His (heterocyclic amino acid, basic amino acid) were selected from a large number of amino acids. Several factors, such as different organic phase, the proportion of organic phase, buffer pH, buffer concentration and flow rate were investigated. As a result, acidic and neutral amino acids showed shorter retention time, higher response value, better peak shape, which were less affected by the chromatographic conditions; and basic amino acids showed longer retention time, lower response values, broader peak. So it was necessary to decrease buffer pH , increase the proportion of aqueous phase, increase the buffer concentration and increase the flow rate for basic amino acids analysis. In addition, adjusting the mobile phase elution gradient, isobaric amino acids can be separated by HILIC column.HLILC-MS/MS method was established for simultaneous determination of acidic (Glu), neutral (Val, Phe) and basic (His, Orn) amino acids in aqueous solution. A single sample run time was 10 min. The calibration curves were linear over the concentration range of 0.150μmol·L-1(r>0.999). The lower limit of quantification (LLOQ) was 0.1μmol·L-1. The lower limit of detections (S/N>3) ranged from 0.01μmol·L-1 to 0.05μmol·L-1. The intra-day and inter-day precisions (RSD) ranged from 0.7 % to 10.3 % and accuracy ranged from 89.1 % to 107.6 %. The recoveries ranged from 91.7 % to 104.5 %. The mixed amino acids standard solution proved to be stable at room temperature for 8 h, at the sample plate for 12 h, at the condition of repeated freezing and thawing for three times and at -80℃freezer for 1 month.As amino acids are the endogenous substances of biological matrix, blank subtraction method was used to establish the standard curves. The calibration was to be conduced by adding up known amounts of amino acids to common plasma to produce a calibration function with an offset according to the endogenous concentration. Blank response values of the endogenous amino acids were subtracted from each calibration point.HLILC-MS/MS method was established for simultaneous determination of acidic (Glu), neutral (Val, Phe) and basic (His, Orn) amino acids in rat plasma. A single sample run time was 10 min. The sample preparation comprised only protein precipitation and the addition of the IS. Blank subtraction method was used to establish the standard curves. The calibration curves were linear over the concentration range of 25800μmol·L-1 (r>0.999) and the lower limit of quantification (LLOQ) was 25μmol·L-1. The intra-day and inter-day precisions (RSD) ranged from 1.5 % to 11.3 % and accuracy ranged from 98.7 % to 110.1 %. The recoveries ranged from 88.7 % to 105.1 %. The quantification results were independent with regard to the biological matrix analyzed. The stability test of rat plasma amino acids showed that Glu, Val and Phe slightly degraded at room temperature for 8 h, at the sample plate for 12 h, at the condition of repeated freezing and thawing for three times. His and Orn proved to be stable.In the second part of the dissertation, the HILIC-MS/MS method was used in the pharmacokinetic study ofα-KGO, which was a drug candidate of anti-radiation.A rapid HLILC-MS/MS method was established for determination of Orn in mouse plasma. A single sample run time was 10 min, and analysis time was 8 min. The sample preparation comprised only protein precipitation and the addition of the IS. Blank subtraction method was used to establish the standard curves. The calibration curves were linear over the concentration range of 254000μmol·L-1(r>0.999) and the lower limit of quantification (LLOQ) was 25μmol·L-1. The intra-day and inter-day precisions (RSD) ranged from 1.3 % to 6.9 % and accuracy ranged from 100.4 % to 102.4 %. The recoveries ranged from 97.3 % to 104.5 %. The quantification results were independent with regard to the biological matrix analyzed. The mouse plasma Orn proved to be stable at room temperature for 8 h, at the sample plate for 12 h, at the condition of repeated freezing and thawing for three times and at -80℃freezer for 2 weeks. The method validation demonstrated that the established method was satisfied with the requirements of pharmacokinetic research.After a single po administration ofα-KGO at two different doses in mice, the time of plasma exogenous Orn concentration reached maximum (Tmax) were 30 and 45 min, respectively. The maximum concentrations (Cmax) of the two groups were 479±89.8μmol·L-1 and 2488.5±304.1μmol·L-1, and statistically significant differences were found among the groups. The area under concentration-time curve (AUC0-t) increased with dose. The terminal phase half-life (t1/2) was about 15 min. The plasma clearance (CL/F) were 85.57 and 38.99 mL·min-1·kg-1, respectively. After a single iv administration ofα-KGO in mice, plasma exogenous Orn concentration reached maximum immediately. The plasma clearance (CL/F) was 46.82 mL·min-1·kg-1. The terminal phase half-life (t1/2) was about 8.5 min. The maximum concentration (Cmax) was 1328.5±63.6μmol·L-1, and statistically significant differences were found compared with the same dose of oral administration. The calculated bioavailability of Orn was 54.7%. |
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