目的: 建立超高效液相色谱-串联质谱法(UHPLC-MS/MS)同时测定4种复方氨基酸注射液中22个氨基酸的含量。方法: 采用Agilent Zorbax SB-C18色谱柱(150 mm ×3.0 mm,5 μm),二元洗脱系统(A相为含0.2%甲酸和0.02%七氟丁酸的水溶液;B相为甲醇)进行梯度洗脱,柱温50 ℃,流速0.4 mL·min-1;采用电喷雾电离正离子模式检测,多反应监测模式测定,运行时间为9.5 min。结果: 甘氨酸(Gly)、L-丙氨酸(Ala)、L-缬氨酸(Val)、L-亮氨酸(Leu)、L-异亮氨酸(Ile)、L-赖氨酸(Lys)、L-甲硫氨酸(Met)、L-组氨酸(His)、L-苯丙氨酸(Phe)、L-精氨酸(Arg)质量浓度在0.32~8 μg·mL-1,L-丝氨酸(Ser)、L-脯氨酸(Pro)、L-苏氨酸(Thr)、L-门冬酰胺(Asn)、L-鸟氨酸(Orn)、L-门冬氨酸(Asp)、L-谷氨酸(Glu)、N-乙酰-L-半胱氨酸(Ace)、L-色氨酸(Trp)、N-乙酰-L-酪氨酸(Aty) 质量浓度在0.16~4 μg·mL-1,L-酪氨酸(Tyr)、L-胱氨酸(Cyss)质量浓度在0.032~0.8 μg·mL-1的范围内线性关系良好(r均>0.99),日内精密度和日间精密度、稳定性、重复性均良好,平均加样回收率为90.0%~101.3%(RSD均≤5.9%,n=6)。应用该方法同时测定了4种复方氨基酸注射液中22个氨基酸含量,各氨基酸测定含量与标识含量的比值为86.67%~102.86%。结论: 该方法操作简便,分析快速,专属性强,灵敏度高,测定结果准确,可用于复方氨基酸注射液中各氨基酸的含量测定,系统全面地进行质量控制及质量评价。
Objective: To establish an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous determination of 22 amino acids contents in 4 kinds of compound amino acid injections. Methods: The gradient elution was achieved on an Agilent Zorbax SB-C18 column (150 mm×3.0 mm, 5 μm) with a binary elution system (phase A, 0.2% formic acid and 0.02% heptafluorobutyric acid in water; phase B, methanol). The column temperature was 50 ℃. The flow rate was 0.4 mL·min-1. The multiple reaction monitoring mode was chosen and detected with an ionization source operating in the positive ionization mode for data acquisition. The run time was 9.5 min. Results: The linear relationship was good for Gly, Ala, Val, Leu, Ile, Lys, Met, His, Phe, and Arg in the concentration ranges of 0.32-8 μg·mL-1, and for Ser, Pro, Thr, Asn, Orn, Asp, Glu, Ace, Trp, and Aty in the concentration ranges of 0.16-4 μg·mL-1, and for Tyr and Cyss in the concentration ranges of 0.032- 0.8 μg·mL-1 (all r >0.99). The intra-day and inter-day precision, the stability, and the repeatability were all good. The average sample recovery rates ranged from 90.0% to 101.3% (all RSD ≤5.9%, n=6). The method was used to simultaneously determine 22 amino acids contents in 4 kinds of compound amino acid injections. The ratios of detection content to identification content of 22 amino acids ranged from 86.67% to 102.86%. Conclusion: The method is simple to operate, rapid in analysis, strong in specificity, high in sensitivity, and accurate in determination results. It can be used to determine each amino acid contents in compound amino acid injections, and systematically and comprehensively carry out quality control and quality evaluation.
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