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Detection of Folic Acid and 5-methyltetrahydrofolate in Serum with Cleanert PAX by LC/MS/MS Method

Application Introduction

Folic acid is a water-soluble vitamin B found and separated in the mid-to-late 1940s.

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Introduction

Folic acid is a water-soluble vitamin B found and separated in the mid-to-late 1940s. It is the general name of the compounds having pteroylglutamic acid molecular structures. In the nature, there are more than 100 kinds of folate derivatives, and the main folate derivative in human plasma is 5-methyltetrahydrofolate. Researches show that folic acid not only is related to megaloblastic anemia, but also is related to neural tube defects, some cancers (such as colon cancer), cardiovascular and cerebrovascular diseases and children's mental retardation. Therefore, rapidly and accurately determining the content of folic acid in human tissue is of great importance to preventing, diagnosing and treating the diseases caused by folic acid. It is very challenging to analyze folic acid due to its poor stability and complicated composition. In this application case, the sample is detected for the contents of folic acid and 5-methyltetrahydrofolate in serum with a Cleanert® PAX 96-well plate for pretreatment and then tested by LC-MS/MS method. The experimental method is very stable and reliable. 

Figure 1 Structure of Folic Acid

Figure 2 Structure of 5-methyltetrahydrofolate

Experimental

Materials

A Cleanert® PAX 96-well plate (30 mg/well), a 96-position nitrogen evaporator, a 96-posiiton positive-pressure device, a 96-well receiving plate and a Unisol C18 (2) (3.0 ´ 100 mm, 3 μm, 110 Å).

Sample Treatment Method

Due to the poor stability of folic acid, it is needed to add antioxidants (vitamin C and 2-mercaptoethanol) during the experiment in order to prevent folic acid from decomposing.

Transfer 400 μL of sample to a 2 mL centrifuge tube, and add 400 μL of aqueous solution to the tube. Keep oscillating the tube for 2 min to mix the solution uniformly, and reserve the solution for purification.

Activation: Add 1 mL of methyl alcohol, 500μL of water and 500μL of aqueous solution containing antioxidants in turn, and depress the mixed solution with a positive-pressure device;

Sample loading: Load 800 μL of the mixed solution, and leave it flow naturally;

Leaching: Add 500 μL of formic acid aqueous solution (concentration: 0.1%) containing antioxidants and 250 μL of methanol solution containing antioxidants in turn, and dry it with the positive-pressure instrument;

Elution: Elute the sample with 500 uL of formic acid-methanol solution (concentration: 2%), and leave the eluant flow naturally;

Dry the eluant with moderate nitrogen at 37 °C, and redissolve it with 100 μL of water. Then carry out sample injection and detection.

Instrumentation

Chromatographic columns: Venusil® MP C18(2) (3.00 ´ 100mm, 3μm, 110Å)

Flow rate: 500 μL/min

Column temperature: 30 °C

Sample size: 10 μL

Mobile phases: A - Formic acid aqueous solution (concentration: 0.1%)

B - Formic acid-methanol solution (concentration: 0.1%)

Scanning mode: ESI normal mode

Collecting mode: Multiple reaction monitoring (MRM)

Table 1 Gradient Conditions

T

A%

B%

0

95

5

1

95

5

1.01

20

80

5

20

80

5.01

95

5

10

95

5

Table 2 Parameter Information

Compound

Retention time/min

Q1

Q3

CE

Folic acid

3.73

442.4

295.1

26

442.4

175.9

56

5-methyltetrahydrofolate

3.54

460.3

313.2

23

460.3

193.6

43

 Results and Discussion

Figure 3 Chromatogram of Standard Solution (20 ng/mL)

Figure 4 Chromatogram of Serum Sample

Figure 5 Chromatogram of Serum Sample with Standard Addition (spiked sample of 20 ng/mL)

Figure 6 Chromatogram of Post-Standard Addition Sample (spiked sample of 20 ng/mL)

In serum, the clinical indexes of folic acid are 6–20.1ng/mL (normal), 3.0-6.0 ng/mL (uncertain) and £ 3.0 ng/mL (clinical deficiency). In order to inspect the method precision and accuracy, take the serum samples with standard addition (concentrations of standard substance added: 1ng/mL, 5 ng/mL and 20 ng/mL) as the methodological inspection samples, and the experimental results are as shown in Table 3. To inspect the matrix effect, the standard addition operation is performed after detection in this experiment, and results show that matrix effect factors are < 9%.

Dilute the sample and determine it again according to the response of the aimed compound. If the deviation between the marked concentration and the calculated value is within ±20%, the minimum quantitative concentrations of folic acid and 5-methyltetrahydrofolate are 0.05 ng/mL and 0.08 ng/mL respectively.

Table 3 Standard Addition Recovery Rate of Serum

Aimed compound

Standard   addition concentration

1ng/mL

Standard   addition concentration 5ng/mL

Standard   addition concentration

20ng/mL

Average recovery rate

(n=5)

RSD

(n=5)

Average recovery rate

(n=5)

RSD

(n=5)

Average recovery rate

(n=5)

RSD

(n=5)

5-methyltetrahydrofolate

89.6%

8.9%

105.8%

5.4%

101.2%

3.3%

Folic acid

91.5%

10.2%

99.4%

3.7%

95.8%

2.3%

Conclusion

In this experiment, folic acid and 5-methyltetrahydrofolate are extracted from serum with Cleanert® PAX. For folic acid and 5-methyltetrahydrofolate, under the standard addition concentration between 1ng/mL and 20 ng/mL, their recovery rates exceed 89%; RSDs are no more than 11% under low standard addition concentration and less than 5% under high standard addition concentration; the minimum quantitative concentrations of folic acid and 5-methyltetrahydrofolate are 0.05 ng/mL and 0.08 ng/mL respectively. Besides, the sample treatment speed greatly improved due to the 96-well positive-pressure device and nitrogen evaporator can fully satisfy the laboratorial detection needs.

  • Cleanert PAX (RP/Strong Anion Exchange)

    It is designed to overcome the limitations of traditional silica based mixed-mode SPE sorbents such as C18/SAX. It is a RP/strong anion exchange mixed-mode polystyrene/ divinylbenzene sorbent, stable from pH 0-14.

  • 96-position Positive Pressure SPE Equipment

    MULTI-SPE M96 is a positive pressure manifold, specially designed for highthroughput sample preparation in research and testing laboratories.

  • Unisol Technology and Column Products

    Bonna-Agela's unique sol-gel process technology generates an uniform surface that produces an universal column applicable to several kinds of HPLC applications.

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