Analysis method of liquid combination of caffeine and its metabolites
A liquid-mass spectrometry and analysis method technology, applied in the field of detection and analysis, can solve the problems of large workload and low efficiency, and achieve the effect of good reliability and high efficiency
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Embodiment 1
[0058] This embodiment provides a liquid chromatography-mass spectrometry analysis method for caffeine and its metabolites, comprising the following steps:
[0059] S1. Determine the parameter conditions for detection and analysis by LC-MS / MS.
[0060] Wherein, the chromatographic conditions are as follows:
[0061] Chromatographic column: adopt ACQMITYHSS T3 column, the specification is i.d.2.1×100mm, 1.8μm.
[0062] Mobile phase: mobile phase A is water containing 0.04% acetic acid, and mobile phase B is acetonitrile containing 0.04% acetic acid. The gradient elution program is shown in Table 1 below:
[0063] Table 1 Gradient elution program table
[0064] time (min) Flow rate (mL / min) A(％) B(％) 0 0.35 95 5 0.5 0.35 95 5 5 0.35 60 40 5.1 0.35 5 95 7.5 0.35 5 95 7.6 0.35 95 5 10 0.35 95 5
[0065] The mass spectrometry conditions are shown in Table 2 and Table 3 below:
[0066] Table 2 Mass Spectrometry ...
Embodiment 2
[0089] Embodiment 2 plasma sample test
[0090] Source of plasma samples: Plasma samples A, B, and C were all from the General Hospital of the Chinese People's Liberation Army (No. 301 Hospital).
[0091] Pre-treatment: Put crushed ice in the centrifuge tube box, take out the plasma sample to be tested from the -80°C refrigerator and put it in the centrifuge tube box with crushed ice to thaw (thaw the sample on ice and all subsequent operations must be done on ice Carry out) until there is no ice cube in the sample, after thawing is complete, vortex for 10s to mix.
[0092] Preparation of the test solution: take 50 μL of the plasma sample to be tested, add 150 μL in methanol solution, 20 μL of the internal standard mixed working solution (100 ng / mL). Vortex for 3min, centrifuge at 12000r / min for 10min at 4°C. After centrifugation, transfer 150 μL of the supernatant to another numbered centrifuge tube, and let stand overnight at -20°C. 12000r / min, then centrifuge at 4°C for ...
Embodiment 3
[0096] Embodiment 3 methodological verification
[0097] (1) Precision
[0098] The same standard solution (50ng / mL) was tested for 3 consecutive days according to the test conditions of Example 1, and one needle was tested every day, and the RSD was calculated. Respectively to the same standard solution (50ng / mL), the test was repeated 3 times at intervals in one day according to the test conditions of Example 1, and the RSD was calculated. The statistical results are shown in Table 9 below:
[0099] Table 9 Statistical Table of Precision Results (N=3)
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[0101]
[0102] (2) Recovery rate
[0103] Adopt plasma sample D (from Chinese People's Liberation Army General Hospital (301 Hospital)) as contrast to test the basic content of 18 kinds of components according to the method of embodiment 2, then add each standard substance respectively, carry out recovery rate test, each sample parallel test 3 Second-rate. The statistics of the results are shown in Table ...
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