Vitamin A is indispensable to the development of multiple human organs and maintenance of human normal physiological function, but its excessive consumption may lead to toxic and side effects; as an antioxidant, vitamin E mainly plays the function of protecting biofilm structures of cells. Recently research shows that vitamin E is also able to regulate gene expression. Although vitamin E deficiency is rare, it is related to newborn dysplasia and inherited metabolic diseases. Therefore it is of great importance to accurately determine the contents of vitamin A and vitamin E in human body. Since vitamin A and vitamin E are easy to oxidize due to their active chemical properties, it is challenging to accurately quantify this kind of substances.
This application case introduces a LC-MS/MS method which purify plasma samples with Cleanert® SLE 96-well plate and extract enriched vitamin A (retinol, VA), α-tocopherol (α-VE) and γ-tocopherol (γ-VE). This experiment method is very stable and reliable, thus it can be used as a solution for VA and VE clinical researches.
Figure 1 VA Structure
Figure 2 α-VE Structure
Figure 3 γ-VE Structure
Cleanert® SLE 96-well plate (200mg/well), Cleanert M96 positive pressure device, Cleanert® V96 N-EVAP, 96-well collecting plate and Bonshell C18 (2.1 ´ 100 mm, 2.7 μm), manufactured by Tianjin Bonna-Agela Technologies Co., Ltd.; LC-MS/MS and API 4000+ (SCIEX); VA, α-VE, γ-VE, d6-α-VE and d4-γ-VE (the above 5 standard substances are purchased from altascientific); d6-VA (purchased from BUCHEM BV); methanol; formic acid; aqueous solution; isooctane; isopropanol; 2, 6-BHT.
Since VA and VE have a poor stability, in order to prevent VA and VE from being oxidized, it is needed to add antioxidant (2, 6-BHT) during experiment.
Transfer 100 μL of plasma to a clean sample vail. Add the internal standard solution, 50 μL of isopropanol and 50 μL of aqueous solution to the vail in turn, and then oscillate the vail on the vortex mixer to mix the substances uniformly.
Sample loading: Load all the premixed solution, and leave it stand still for 5min;
Elution: Respectively elute the samples with 2mL of isooctane/isopropanol (9/1, containing 1g BHT/L) for 4 times (500 μL/time), and combine the eluant; after the last elution, place the Cleanert® SLE and collection plate on the Cleanert M96 positive pressure device as a whole, and regulate the apparatus pressure to 0.02 MPa. About 30s later, the eluant will be completely collected.
Dry the eluant with Cleanert V96 N-EVAP in moderate nitrogen mode at ambient temperature, redissolve it with 500 μL of formic acid methanol (concentration: 0.1%, containing 1 g BHT/L), and then detect the sample.
Chromatographic columns: Bonshell® C18 (2.1 ´ 100 mm, 2.7 μm)
Flow rate: 200 μL/min
Column temperature: 30 °C
Sample size: 5 μL
Mobile phase condition: 100% organic phase (0.1% formic acid methanol) equi-separation for 6 min
Scanning mode: Normal electrospray ionization mode
Collecting mode: Multiple-reaction monitoring (MRM)
Table 1 Parameters
Results and Discussion
Relation between linear range and sensitivity
The standard curve concentration range is determined according to the content of aimed compound in human body. During the experiment, prepare 7 samples with different concentrations with the plasma sample free of the aimed compound at calibration curve points: VA concentrations: 0.05, 0.15, 0.3, 0.5, 0.8, 1, 5 μg/mL; α-VE concentrations: 1, 2, 4, 8, 10, 20, 25 μg/mL; γ-VE concentrations: 0.2, 0.5, 1, 1.5, 3, 4.5, 8 μg/mL; purify the obtained samples by the aforesaid sample pretreatment method and detect them. See Table 2 for the detection results.
Table 2 Relation between Linear Range and Sensitivity
Correlation coefficient (R2)
Method Quantitation Limit (MQL) (μg/mL)
y = 1.3073x + 0.5242
y = 1.5237x + 0.1241
y = 2.744x + 0.0425
Relation between method precision and accuracy
In order to research the relation between method precision and accuracy, separate 3 samples with low, medium and high concentrations from each compound, and treat each above sample in parallel to 6 samples. See Table 3 for the obtained data.
Table 3 Relation between Method Precision and Accuracy
Standard addition concentration
Average recovery rate
Test the samples purified with Cleanert® SLE as per the aforesaid LC-MS/MS testing conditions, and the obtained chromatogram is as shown in Figure 4-6.
The liquid tandem matrix effect is mainly caused by the large amount of phospholipid in plasma samples. In order to exploit the capability of Cleanert® SLE technology in phospholipid removal, sample two pairs of phospholipid characteristic ions for detection at the same time: 496/184 and 524/184. The obtained chromatogram is as shown in Figure 7.
Figure 4 Chromatogram of VA Plasma Added with Standard Solution (concentration: 0.15 μg/mL)
Figure 5 Chromatogram of α-VE Plasma Added with Standard Solution (concentration: 2 μg/mL)
Figure 6 Chromatogram of γ-VE Plasma Added with Standard Solution (concentration: 0.5 μg/mL)
Figure 7 Chromatogram of Plasma Added with Standard Phospholipid Sample
This application introduces a simple but efficient sample pretreatment method, that is, purify plasma samples and extract enriched liposoluble VA and VE in high throughput with Cleanert SLE 96-well plate. This experiment method is easy to operate and stable, thus it can be used for substituting the time-consuming and inefficient liquid-liquid extraction technology in clinical research and drug metabolism field.