Method for simultaneously determining fat-soluble vitamins and carotenoids in serum

Abstract

A method for simultaneously determining fat-soluble vitamins and carotenoids in serum, and belonging to the technical field of analytical chemistry, includes an ionic liquid (IL) or a binary mixed solvent composed of an IL and another solvent is adopted as an extractant; the biological samples are pre-treated by liquid-liquid extraction (LLE) and then detected by high-performance liquid chromatography (HPLC); retinyl acetate and trans-β-apo-8′-carotenal are adopted as the internal standards for fat-soluble vitamins and carotenoids, respectively; and the internal standard method is adopted to establish standard curves for quantification based on the retention time and the UV-Vis absorption spectrum. Compared with the existing methods, in the disclosure, the pretreatment process is simple and easy to be conducted, the sample can be prepared in a short time, and the toxic and harmful organic solvent is used at a reduced amount.

Claims

1. A method for simultaneously determining fat-soluble vitamins and carotenoids in serum, comprising: with an ionic liquid (IL) or a binary mixed solvent composed of an IL and an organic solvent as an extractant, pretreating serum by liquid-liquid extraction (LLE); and with retinyl acetate and trans-β-apo-8′-carotenal as internal standards for fat-soluble vitamins and carotenoids, respectively, subjecting the obtained IL extract phase to high-performance liquid chromatography (HPLC) to determine the contents of fat-soluble vitamins and carotenoids in serum.

2. The method for simultaneously determining fat-soluble vitamins and carotenoids in serum according to claim 1, wherein, the IL is composed of two parts: cation M.sup.+ and anion N.sup.−; the cation M.sup.+ is one of imidazole, pyridine, piperidine, pyrrolidine, amine and phosphoric acid cations with one or more substituents, and the substituent is alkyl, alkenyl or aryl; and the anion N.sup.− is one of hydrophobic anions such as tetrafluoroborate, hexafluoroborate and bis(trifluoromethanesulfonyl)imide.

3. The method for simultaneously determining fat-soluble vitamins and carotenoids in serum according to claim 1, wherein, the IL has a mole percent of 1% to 90% in the binary mixed solvent.

4. The method for simultaneously determining fat-soluble vitamins and carotenoids in serum according to claim 1, wherein, the organic solvent is dichloromethane (DCM), chloroform, dichloroethane, ethyl acetate, n-butanol, n-heptanol, n-octanol, n-pentane, n-hexane, n-heptane, n-octane, cyclopentane, cyclohexane, petroleum ether with a boiling range of 60° C. to 90° C., petroleum ether with a boiling range of 90° C. to 120° C., or toluene.

5. The method for simultaneously determining fat-soluble vitamins and carotenoids in serum according to claim 1, wherein, the serum is pretreated by LLE, with 50 μL to 500 μL of extractant for 250 μL of serum.

6. The method for simultaneously determining fat-soluble vitamins and carotenoids in serum according to claim 1, wherein, the LLE recovery rate for fat-soluble vitamins and carotenoids is 50% to 95%.

7. The method for simultaneously determining fat-soluble vitamins and carotenoids in serum according to claim 1, wherein, the HPLC analysis is conducted at the following conditions: stationary phase: YMC C30 chromatographic column, 4.6 mm×250 mm, 5 μm; mobile phase A: a methanol aqueous solution with a volume fraction of more than 90%; mobile phase B: a mixed solution of i-propanol/n-hexane with a volume fraction of more than 30%; elution mode: gradient elution, with a flow rate of 1 mL/min; detection wavelength: 324 nm for vitamin A; 263 nm for vitamin D; 290 nm for tocopherol; 254 nm for vitamin K; 450 nm for carotenoid; column temperature: 25° C., and injection volume: 10 μL.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIGS. 1A-1O are schematic diagrams of the molecular structures of fat-soluble vitamins and carotenoids;

(2) FIG. 2 shows the comparison of the serum sample pretreatment process in the disclosure with the traditional serum sample pretreatment process; and

(3) FIGS. 3A-3D shows liquid chromatograms for typical fat-soluble vitamins and carotenoids in serum, where, (a) is for vitamin A; (b) is for 25-hydroxyvitamin D.sub.3; (c) is for γ-tocopherol; (d) is for lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene and lycopene.

DETAILED DESCRIPTION

(4) To make persons skilled in the art better understand the solutions of the disclosure, the following clearly and completely describes the technical solutions in the examples of the disclosure with reference to the accompanying drawings in the examples of the disclosure. Apparently, the described examples are merely some rather than all of the examples of the disclosure. All other examples obtained by a person of ordinary skill in the art based on the examples of the disclosure without creative efforts shall fall within the protection scope of the disclosure.

(5) Moreover, the terms “include”, “comprise”, and any other variants mean to cover the non-exclusive inclusion, for example, a process, method, system, product, or device that includes a list of steps or units is not necessarily limited to those steps or units which are clearly listed, but may include other steps or units which are not expressly listed or inherent to such a process, method, system, product, or device.

(6) The disclosure will be further explained in detail below with reference to the accompanying drawings.

(7) The method for simultaneously determining fat-soluble vitamins and carotenoids in serum disclosed in the disclosure includes the following steps:

(8) 1) a solution of retinyl acetate and trans-β-apo-8′-carotenal in methanol with a known concentration is added to the to-be-tested serum, and the resulting mixture is well mixed;

(9) 2) then an extractant is added to the to-be-tested serum, the resulting mixture is vortexed for 2 min and then centrifuged in a 4° C. centrifuge at 12,000 r/min for 10 min, and the IL extract phase is collected;

(10) 3) a certain volume of each of stock solutions for fat-soluble vitamin and carotenoid standards is taken and then diluted with simulated serum to give standard solutions with a series of concentrations, and the standard solutions are treated according to steps 1) and 2) to give the extract phases of the standards; and

(11) 4) the serum extract phase and the standard extract phases are analyzed by LC, the internal standard method is adopted to establish standard curves for fat-soluble vitamins and carotenoids, and the peak area ratio of each to-be-tested component in serum to the internal standard is compared with the standard curve to obtain the content of each fat-soluble nutrient in serum.

(12) The fat-soluble vitamins mainly include vitamin A, vitamin D (vitamin D.sub.2, vitamin D.sub.3, and metabolites of 25-hydroxyvitamin D.sub.3 and 25-hydroxyvitamin D.sub.2), vitamin E (α-tocopherol and γ-tocopherol), and vitamin K (vitamin K.sub.1 and vitamin K.sub.2) (FIGS. 1A-1I). The carotenoids mainly include lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene, lycopene, etc. (FIGS. 1J-1O).

(13) As shown in FIG. 2, compared with the existing methods, the method of the disclosure has the following advantages: The pretreatment process is simple and easy to be conducted, the sample can be prepared in a short time, and the toxic and harmful organic solvent is used at a reduced amount. The HPLC analysis method exhibits an excellent separation effect and a high detection accuracy, and can achieve the simultaneous detection of more than ten fat-soluble nutrients within 40 min (the HPLC chromatograms of typical fat-soluble vitamins and carotenoids in serum are shown in FIGS. 3A-3D). Moreover, with high-throughput and low-cost, the disclosure can be easily generalized in clinics.

Example 1

(14) A method for simultaneously determining fat-soluble vitamins and carotenoids in serum included the following steps:

(15) 1) 5 uL of a solution of retinyl acetate and trans-β-apo-8′-carotenal in methanol with a known concentration was added to 250 uL of serum, and the resulting mixture was well mixed;

(16) 2) then 100 uL of a solution of 1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide ([bmim][Tf.sub.2N]) in n-hexane with a mole fraction of 50% was added to the serum, the resulting mixture was vortexed for 2 min and then centrifuged in a 4° C. centrifuge at 12,000 r/min for 10 min, and the TL extract phase was collected;

(17) 3) a certain volume of each of stock solutions for fat-soluble vitamin and carotenoid standards was taken and then diluted with simulated serum to give standard solutions with a series of concentrations, and 250 uL of each of the standard solutions was taken and treated according to steps 1) and 2) to give the extract phases of the standards; and

(18) 4) the serum extract phase and the standard extract phases were analyzed by LC, the internal standard method was adopted to establish standard curves for fat-soluble vitamins and carotenoids, and the peak area ratio of each to-be-tested component in serum to the internal standard was compared with the standard curve to obtain the content of each fat-soluble nutrient in serum. The HPLC analysis was conducted at the following conditions: chromatographic column: YMC C30 (4.6 mm×250 mm, 5 m); mobile phase A: methanol; mobile phase B: a mixed solution of i-propanol/n-hexane with a volume fraction of 30%; flow rate: 1 mL/min; elution mode: gradient elution (0 min to 20 min, 100% A to 50% A; 20 min to 22 min, 50% A to 0% A; 22 min to 30 min, 0% A to 0% A; 30 min to 31 min, 0% A to 100% A); multi-wavelength detection (324 nm for vitamin A; 263 nm for vitamin D; 290 nm for vitamin E; 254 nm for vitamin K; and 450 nm for carotenoid); column temperature: 25° C., and injection volume: 10 μL.

(19) This quantitative analysis method is accurate and reliable. The sample recovery rate of the detected fat-soluble vitamins and carotenoids is 83.2% to 108.5%, and the minimum detection limit is 0.005 ug/mL to 0.025 ug/mL. As detected by the experiment, the contents of vitamin A, 25-hydroxyvitamin D.sub.3, α-tocopherol, γ-tocopherol, lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene and lycopene in the serum of 20 normal people are 1.05±0.23 μmol/L, 0.051±0.036 μmol/L, 34.2±10.8 μmol/L, 4.14±0.9 μmol/L, 0.48±0.24 μmol/L, 0.072±0.026 μmol/L, 0.16±0.14 μmol/L, 0.073±0.057 μmol/L, 0.53±0.37 μmol/L, and 0.17±0.12 μmol/L, respectively, which are consistent with the content ranges of these nutrients in a normal person reported in references.

Example 2

(20) A method for simultaneously determining fat-soluble vitamins and carotenoids in serum included the following steps:

(21) 1) 15 uL of a solution of retinyl acetate and trans-β-apo-8′-carotenal in methanol with a known concentration was added to 250 uL of serum, and the resulting mixture was well mixed;

(22) 2) then 50 uL of a solution of 1-hexyl-3-methylimidazolium tetrafluoroborate ([hmim][BF.sub.4]) in DCM with a mole fraction of 30% was added to the serum, the resulting mixture was vortexed for 2 min and then centrifuged in a 4° C. centrifuge at 12,000 r/min for 10 min, and the L extract phase was collected;

(23) 3) a certain volume of each of stock solutions for fat-soluble vitamin and carotenoid standards was taken and then diluted with simulated serum to give standard solutions with a series of concentrations, and 250 uL of each of the standard solutions was taken and treated according to steps 1) and 2) to give the extract phases of the standards; and

(24) 4) the serum extract phase and the standard extract phases were analyzed by LC, the internal standard method was adopted to establish standard curves for fat-soluble vitamins and carotenoids, and the peak area ratio of each to-be-tested component in serum to the internal standard was compared with the standard curve to obtain the content of each fat-soluble nutrient in serum.

(25) The HPLC analysis was conducted at the following conditions: chromatographic column: YMC C30; mobile phase A: a methanol aqueous solution with a volume fraction of 90%; mobile phase B: a mixed solution of i-propanol/n-hexane with a volume fraction of 40%; flow rate: 1 mL/min; elution mode: gradient elution (0 min to 15 min, 100% A to 40% A; 15 min to 18 min, 40% A to 0% A; 18 min to 23 min, 0% A to 0% A; 23 min to 25 min, 0% A to 100% A); multi-wavelength detection (324 nm for vitamin A; 263 nm for vitamin D; 290 nm for vitamin E; 254 nm for vitamin K; and 450 nm for carotenoid); column temperature: 25° C., and injection volume: 10 μL.

(26) This quantitative analysis method is accurate and reliable. The sample recovery rate of the detected fat-soluble vitamins and carotenoids is 89.8% to 110.2%, and the minimum detection limit is 0.005 ug/mL to 0.025 ug/mL. As detected by the experiment, the contents of vitamin A, 25-hydroxyvitamin D.sub.3, α-tocopherol, γ-tocopherol, lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene and lycopene in the serum of 30 normal people are 2.45±0.53 μmol/L, 0.068±0.04 μmol/L, 39.1±14.7 μmol/L, 2.96±0.77 μmol/L, 0.64±0.31 μmol/L, 0.13±0.09 μmol/L, 0.089±0.067 μmol/L, 0.046±0.033 μmol/L, 0.44±0.34 μmol/L, and 0.21±0.14 μmol/L, respectively, which are consistent with the content ranges of these nutrients in a normal person reported in references.

Example 3

(27) A method for simultaneously determining fat-soluble vitamins and carotenoids in serum included the following steps:

(28) 1) 10 uL of a solution of retinyl acetate and trans-β-apo-8′-carotenal in methanol with a known concentration was added to 250 uL of serum, and the resulting mixture was well mixed;

(29) 2) then 200 uL of a solution of N-dodecylpyridinium hexafluorophosphate ([C.sub.12Py][PF.sub.6]) in toluene with a mole fraction of 40% was added to the serum, the resulting mixture was vortexed for 2 min and then centrifuged in a 4° C. centrifuge at 12,000 r/min for 10 min, and the L extract phase was collected;

(30) 3) a certain volume of each of stock solutions for fat-soluble vitamin and carotenoid standards was taken and then diluted with simulated serum to give standard solutions with a series of concentrations, and 250 uL of each of the standard solutions was taken and treated according to steps 1) and 2) to give the L extract phases of the standards; and

(31) 4) the serum extract phase and the standard extract phases were analyzed by LC, the internal standard method was adopted to establish standard curves for fat-soluble vitamins and carotenoids, and the peak area ratio of each to-be-tested component in serum to the internal standard was compared with the standard curve to obtain the content of each fat-soluble nutrient in serum.

(32) The HPLC analysis was conducted at the following conditions: chromatographic column: YMC C30; mobile phase A: a methanol aqueous solution with a volume fraction of 95%; mobile phase B: a mixed solution of i-propanol/n-hexane with a volume fraction of 50%; flow rate: 1 mL/min; elution mode: gradient elution (0 min to 30 min, 100% A to 10% A; 30 min to 32 min, 10% A to 10% A; 32 min to 33 min, 10% A to 100% A); multi-wavelength detection (324 nm for vitamin A; 263 nm for vitamin D; 290 nm for vitamin E; 254 nm for vitamin K; and 450 nm for carotenoid); column temperature: 25° C., and injection volume: 10 μL.

(33) This quantitative analysis method is accurate and reliable. The sample recovery rate of the detected fat-soluble vitamins and carotenoids is 90.3% to 99.5%, and the minimum detection limit is 0.005 ug/mL to 0.025 ug/mL. As detected by the experiment, the contents of vitamin A, 25-hydroxyvitamin D.sub.3, α-tocopherol, γ-tocopherol, lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene and lycopene in the serum of 50 normal people are 1.88±0.43 μmol/L, 0.13±0.08 μmol/L, 38.9±14.2 μmol/L, 5.66±1.7 μmol/L, 0.61±0.16 μmol/L, 0.089±0.02 μmol/L, 0.14±0.03 μmol/L, 0.038±0.017 μmol/L, 0.62±0.45 μmol/L, and 0.19±0.13 μmol/L, respectively, which are consistent with the content ranges of these nutrients in a normal person reported in references.

Example 4

(34) A method for simultaneously determining fat-soluble vitamins and carotenoids in serum included the following steps:

(35) 1) 20 uL of a solution of retinyl acetate and trans-β-apo-8′-carotenal in methanol with a known concentration was added to 250 uL of serum, and the resulting mixture was well mixed;

(36) 2) then 150 uL of a solution of 1-benzyl-3-methylimidazolium hexafluorophosphate ([bzmim][PF.sub.6]) in cyclopentane with a mole fraction of 70% was added to the serum, the resulting mixture was vortexed for 2 min and then centrifuged in a 4° C. centrifuge at 12,000 r/min for 10 min, and the TL extract phase was collected;

(37) 3) a certain volume of each of methanol stock solutions for fat-soluble vitamin and carotenoid standards was taken and then diluted with simulated serum to give standard solutions with a series of concentrations, and 250 uL of each of the standard solutions was taken and treated according to steps 1) and 2) to give the IL extract phases of the standards; and

(38) 4) the serum extract phase and the standard extract phases were analyzed by LC, the internal standard method was adopted to establish standard curves for fat-soluble vitamins and carotenoids, and the peak area ratio of each to-be-tested component in serum to the internal standard was compared with the standard curve to obtain the content of each fat-soluble nutrient in serum.

(39) The HPLC analysis was conducted at the following conditions: chromatographic column: YMC C30; mobile phase A: a methanol aqueous solution with a volume fraction of 98%; mobile phase B: a mixed solution of i-propanol/n-hexane with a volume ratio of 7/3; flow rate: 1 mL/min; elution mode: gradient elution (0 min to 25 min, 100% A to 0% A; 25 min to 35 min, 0% A to 100% A); multi-wavelength detection (324 nm for vitamin A; 263 nm for vitamin D; 290 nm for vitamin E; 254 nm for vitamin K; and 450 nm for carotenoid); column temperature: 25° C., and injection volume: 10 μL.

(40) This quantitative analysis method is accurate and reliable. The sample recovery rate of the detected fat-soluble vitamins and carotenoids is 86.5% to 112.3%, and the minimum detection limit is 0.005 ug/mL to 0.025 ug/mL. As detected by the experiment, the contents of vitamin A, 25-hydroxyvitamin D.sub.3, α-tocopherol, γ-tocopherol, lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene and lycopene in the serum of 40 normal people are 0.75±0.26 μmol/L, 0.098±0.03 μmol/L, 33.3±11.1 μmol/L, 6.23±0.61 μmol/L, 0.70±0.34 μmol/L, 0.065±0.022 μmol/L, 0.12±0.05 μmol/L, 0.039±0.015 μmol/L, 0.59±0.31 μmol/L, and 0.11±0.092 μmol/L, respectively, which are consistent with the content ranges of these nutrients in a normal person reported in references.

Example 5

(41) A method for simultaneously determining fat-soluble vitamins and carotenoids in serum included the following steps:

(42) 1) 15 uL of a solution of retinyl acetate and trans-β-apo-8′-carotenal in methanol with a known concentration was added to 250 uL of serum, and the resulting mixture was well mixed;

(43) 2) then 400 uL of a solution of N-octyl-N-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide ([OPyr][Tf.sub.2N) in ethyl acetate with a mole fraction of 10% was added to the serum, the resulting mixture was vortexed for 2 min and then centrifuged in a 4° C. centrifuge at 12,000 r/min for 10 min, and the IL extract phase was collected;

(44) 3) a certain volume of each of methanol stock solutions for fat-soluble vitamin and carotenoid standards was taken and then diluted with simulated serum to give standard solutions with a series of concentrations, and 250 uL of each of the standard solutions was taken and treated according to steps 1) and 2) to give the IL extract phases of the standards; and

(45) 4) the serum extract phase and the standard extract phases were analyzed by LC, the internal standard method was adopted to establish standard curves for fat-soluble vitamins and carotenoids, and the peak area ratio of each to-be-tested component in serum to the internal standard was compared with the standard curve to obtain the content of each fat-soluble nutrient in serum.

(46) The HPLC analysis was conducted at the following conditions: chromatographic column: YMC C30; mobile phase A: methanol; mobile phase B: a mixed solution of i-propanol/n-hexane with a volume fraction of 60%; flow rate: 1 mL/min; elution mode: gradient elution (0 min to 10 min, 100% A to 85% A; 10 min to 25 min, 85% A to 0% A; 25 min to 30 min, 0% A to 100% A); multi-wavelength detection (324 nm for vitamin A; 263 nm for vitamin D; 290 nm for vitamin E; 254 nm for vitamin K; and 450 nm for carotenoid); column temperature: 25° C., and injection volume: 10 μL.

(47) This quantitative analysis method is accurate and reliable. The sample recovery rate of the detected fat-soluble vitamins and carotenoids is 80.3% to 98.2%, and the minimum detection limit is 0.005 ug/mL to 0.025 ug/mL. As detected by the experiment, the contents of vitamin A, 25-hydroxyvitamin D.sub.3, α-tocopherol, γ-tocopherol, lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene and lycopene in the serum of 60 normal people are 2.58±0.70 μmol/L, 0.034±0.026 μmol/L, 35.2±4.8 μmol/L, 3.14±0.6 μmol/L, 0.62±0.18 μmol/L, 0.042±0.017 μmol/L, 0.10±0.09 μmol/L, 0.044±0.033 μmol/L, 0.59±0.31 μmol/L, and 0.11±0.06 μmol/L, respectively, which are consistent with the content ranges of these nutrients in a normal person reported in references.

Example 6

(48) A method for simultaneously determining fat-soluble vitamins and carotenoids in serum included the following steps:

(49) 1) 5 uL of a solution of retinyl acetate and trans-β-apo-8′-carotenal in methanol with a known concentration was added to 250 uL of serum, and the resulting mixture was well mixed;

(50) 2) then 100 uL of a solution of tetrabutylammonium bis(trifluoromethylsulfonyl)imide ([N.sub.4,4,4,4][Tf.sub.2N]) in n-octanol with a mole fraction of 60% was added to the serum, the resulting mixture was vortexed for 2 min and then centrifuged in a 4° C. centrifuge at 12,000 r/min for 10 min, and the IL extract phase was collected;

(51) 3) a certain volume of each of stock solutions for fat-soluble vitamin and carotenoid standards was taken and then diluted with simulated serum to give standard solutions with a series of concentrations, and 250 uL of each of the standard solutions was taken and treated according to steps 1) and 2) to give the IL extract phases of the standards; and

(52) 4) the serum extract phase and the standard extract phases were analyzed by LC, the internal standard method was adopted to establish standard curves for fat-soluble vitamins and carotenoids, and the peak area ratio of each to-be-tested component in serum to the internal standard was compared with the standard curve to obtain the content of each fat-soluble nutrient in serum.

(53) The HPLC analysis was conducted at the following conditions: chromatographic column: YMC C30; mobile phase A: a methanol aqueous solution with a volume fraction of 98%; mobile phase B: i-propanol; flow rate: 1 mL/min; elution mode: gradient elution (0 min to 10 min, 100% A to 70% A; 10 min to 20 min, 70% A to 0% A; 20 min to 30 min, 0% A to 100% A); multi-wavelength detection (324 nm for vitamin A; 263 nm for vitamin D; 290 nm for vitamin E; 254 nm for vitamin K; and 450 nm for carotenoid); column temperature: 25° C., and injection volume: 10 μL.

(54) This quantitative analysis method is accurate and reliable. The sample recovery rate of the detected fat-soluble vitamins and carotenoids is 93.2% to 105.8%, and the minimum detection limit is 0.005 ug/mL to 0.025 ug/mL. As detected by the experiment, the contents of vitamin A, 25-hydroxyvitamin D.sub.3, α-tocopherol, γ-tocopherol, lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene and lycopene in the serum of 35 normal people are 2.05±0.66 μmol/L, 0.048±0.04 μmol/L, 35.2±7.8 μmol/L, 5.06±1.0 μmol/L, 0.46±0.12 μmol/L, 0.063±0.039 μmol/L, 0.12±0.05 μmol/L, 0.030±0.019 μmol/L, 0.44±0.19 μmol/L, and 0.43±0.25 μmol/L, respectively, which are consistent with the content ranges of these nutrients in a normal person reported in references.

Example 7

(55) A method for simultaneously determining fat-soluble vitamins and carotenoids in serum included the following steps:

(56) 1) 20 uL of a solution of retinyl acetate and trans-β-apo-8′-carotenal in methanol with a known concentration was added to 250 uL of serum, and the resulting mixture was well mixed;

(57) 2) then 80 uL of a solution of N-hexadecyl-N-methylpiperidinium hexafluorophosphate ([C.sub.16MPip][PF.sub.6]) in petroleum ether (boiling range: 60° C. to 90° C.) with a mole fraction of 50% was added to the serum, the resulting mixture was vortexed for 2 min and then centrifuged in a 4° C. centrifuge at 12,000 r/min for 10 min, and the IL extract phase was collected;

(58) 3) a certain volume of each of stock solutions for fat-soluble vitamin and carotenoid standards was taken and then diluted with simulated serum to give standard solutions with a series of concentrations, and 250 uL of each of the standard solutions was taken and treated according to steps 1) and 2) to give the L extract phases of the standards; and

(59) 4) the serum extract phase and the standard extract phases were analyzed by LC, the internal standard method was adopted to establish standard curves for fat-soluble vitamins and carotenoids, and the peak area ratio of each to-be-tested component in serum to the internal standard was compared with the standard curve to obtain the content of each fat-soluble nutrient in serum.

(60) The HPLC analysis was conducted at the following conditions: chromatographic column: YMC C30; mobile phase A: a methanol aqueous solution with a volume fraction of 93%; mobile phase B: a mixed solution of i-propanol/n-hexane with a volume fraction of 80%; flow rate: 1 mL/min; elution mode: gradient elution (0 min to 20 min, 100% A to 50% A; 20 min to 23 min, 50% A to 0% A; 23 min to 30 min, 0% A to 100% A); multi-wavelength detection (324 nm for vitamin A; 263 nm for vitamin D; 290 nm for vitamin E; 254 nm for vitamin K; and 450 nm for carotenoid); column temperature: 25° C., and injection volume: 10 μL.

(61) This quantitative analysis method is accurate and reliable. The sample recovery rate of the detected fat-soluble vitamins and carotenoids is 90.4% to 113.5%, and the minimum detection limit is 0.005 ug/mL to 0.025 ug/mL. As detected by the experiment, the contents of vitamin A, 25-hydroxyvitamin D.sub.3, α-tocopherol, γ-tocopherol, lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene and lycopene in the serum of 40 normal people are 1.63±0.35 μmol/L, 0.058±0.045 μmol/L, 30.2±8.2 μmol/L, 3.45±1.2 μmol/L, 0.61±0.16 μmol/L, 0.067±0.036 μmol/L, 0.14±0.03 μmol/L, 0.06±0.06 μmol/L, 0.62±0.45 μmol/L, and 0.07±0.03 μmol/L, respectively, which are consistent with the content ranges of these nutrients in a normal person reported in references.

Example 8

(62) A method for simultaneously determining fat-soluble vitamins and carotenoids in serum included the following steps:

(63) 1) 15 uL of a solution of retinyl acetate and trans-β-apo-8′-carotenal in methanol with a known concentration was added to 250 uL of serum, and the resulting mixture was well mixed;

(64) 2) then 120 uL of a solution of N-octylpyridinium tetrafluoroborate ([OPy][BF.sub.4) in cyclohexane with a mole fraction of 10% was added to the serum, the resulting mixture was vortexed for 2 min and then centrifuged in a 4° C. centrifuge at 12,000 r/min for 10 min, and the L extract phase was collected;

(65) 3) a certain volume of each of stock solutions for fat-soluble vitamin and carotenoid standards was taken and then diluted with simulated serum to give standard solutions with a series of concentrations, and 250 uL of each of the standard solutions was taken and treated according to steps 1) and 2) to give the IL extract phases of the standards; and

(66) 4) the serum extract phase and the standard extract phases were analyzed by LC, the internal standard method was adopted to establish standard curves for fat-soluble vitamins and carotenoids, and the peak area ratio of each to-be-tested component in serum to the internal standard was compared with the standard curve to obtain the content of each fat-soluble nutrient in serum.

(67) The HPLC analysis was conducted at the following conditions: chromatographic column: YMC C30; mobile phase A: a methanol aqueous solution with a volume fraction of 97%; mobile phase B: a mixed solution of i-propanol/n-hexane with a volume fraction of 90%; flow rate: 1 mL/min; elution mode: gradient elution (0 min to 18 min, 100% A to 40% A; 18 min to 25 min, 40% A to 0% A; 25 min to 33 min, 0% A to 100% A); multi-wavelength detection (324 nm for vitamin A; 263 nm for vitamin D; 290 nm for vitamin E; 254 nm for vitamin K; and 450 nm for carotenoid); column temperature: 25° C., and injection volume: 10 μL.

(68) This quantitative analysis method is accurate and reliable. The sample recovery rate of the detected fat-soluble vitamins and carotenoids is 84.3% to 97.5%, and the minimum detection limit is 0.005 ug/mL to 0.025 ug/mL. As detected by the experiment, the contents of vitamin A, 25-hydroxyvitamin D.sub.3, α-tocopherol, γ-tocopherol, lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene and lycopene in the serum of 60 normal people are 1.91±0.12 μmol/L, 0.068±0.04 mol/L, 35.6±13.1 μmol/L, 3.28±1.7 μmol/L, 0.46±0.12 μmol/L, 0.042±0.023 μmol/L, 0.21±0.05 μmol/L, 0.063±0.042 μmol/L, 0.85±0.27 μmol/L, and 0.28±0.09 μmol/L, respectively, which are consistent with the content ranges of these nutrients in a normal person reported in references.

Example 9

(69) A method for simultaneously determining fat-soluble vitamins and carotenoids in serum included the following steps:

(70) 1) 10 uL of a solution of retinyl acetate and trans-β-apo-8′-carotenal in methanol with a known concentration was added to 250 uL of serum, and the resulting mixture was well mixed;

(71) 2) then 500 uL of a solution of hexadecyl trimethylphosphonium hexafluorophosphate ([P1,1,1,16][PF.sub.6]) in n-heptanol with a mole fraction of 20% was added to the serum, the resulting mixture was vortexed for 2 min and then centrifuged in a 4° C. centrifuge at 12,000 r/min for 10 min, and the TL extract phase was collected;

(72) 3) a certain volume of each of stock solutions for fat-soluble vitamin and carotenoid standards was taken and then diluted with simulated serum to give standard solutions with a series of concentrations, and 250 uL of each of the standard solutions was taken and treated according to steps 1) and 2) to give the IL extract phases of the standards; and

(73) 4) the serum extract phase and the standard extract phases were analyzed by LC, the internal standard method was adopted to establish standard curves for fat-soluble vitamins and carotenoids, and the peak area ratio of each to-be-tested component in serum to the internal standard was compared with the standard curve to obtain the content of each fat-soluble nutrient in serum.

(74) The IPLC analysis was conducted at the following conditions: chromatographic column: YMC C30; mobile phase A: a methanol aqueous solution with a volume fraction of 96%; mobile phase B: a mixed solution of i-propanol/n-hexane with a volume fraction of 65%; flow rate: 1 mL/min; elution mode: gradient elution (0 min to 25 min, 100% A to 0% A; 25 min to 28 min, 0% A to 100% A); multi-wavelength detection (324 nm for vitamin A; 263 nm for vitamin D; 290 nm for vitamin E; 254 nm for vitamin K; and 450 nm for carotenoid); column temperature: 25° C., and injection volume: 10 μL.

(75) This quantitative analysis method is accurate and reliable. The sample recovery rate of the detected fat-soluble vitamins and carotenoids is 85.4% to 100.3%, and the minimum detection limit is 0.005 ug/mL to 0.025 ug/mL. As detected by the experiment, the contents of vitamin A, 25-hydroxyvitamin D.sub.3, α-tocopherol, γ-tocopherol, lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene and lycopene in the serum of 70 normal people are 3.41±0.53 μmol/L, 0.081±0.04 μmol/L, 44.2±12.6 μmol/L, 7.14±2.45 μmol/L, 0.33±0.14 μmol/L, 0.052±0.046 μmol/L, 0.13±0.08 μmol/L, 0.062±0.037 μmol/L, 0.72±0.35 μmol/L, and 0.32±0.04 μmol/L, respectively, which are consistent with the content ranges of these nutrients in a normal person reported in references.

Example 10

(76) A method for simultaneously determining fat-soluble vitamins and carotenoids in serum included the following steps:

(77) 1) 20 uL of a solution of retinyl acetate and trans-β-apo-8′-carotenal in methanol with a known concentration was added to 250 uL of serum, and the resulting mixture was well mixed;

(78) 2) then 200 uL of a solution of N-butyl-N-methylpiperidinium bis(trifluoromethylsulfonyl)imide ([BMPip][Tf.sub.2N]) in dichloroethane with a mole fraction of 35% was added to the serum, the resulting mixture was vortexed for 2 min and then centrifuged in a 4° C. centrifuge at 12,000 r/min for 10 min, and the IL extract phase was collected;

(79) 3) a certain volume of each of stock solutions for fat-soluble vitamin and carotenoid standards was taken and then diluted with simulated serum to give standard solutions with a series of concentrations, and 250 uL of each of the standard solutions was taken and treated according to steps 1) and 2) to give the IL extract phases of the standards; and

(80) 4) the serum extract phase and the standard extract phases were analyzed by LC, the internal standard method was adopted to establish standard curves for fat-soluble vitamins and carotenoids, and the peak area ratio of each to-be-tested component in serum to the internal standard was compared with the standard curve to obtain the content of each fat-soluble nutrient in serum.

(81) The HPLC analysis was conducted at the following conditions: chromatographic column: YMC C30; mobile phase A: methanol; mobile phase B: a mixed solution of i-propanol/n-hexane with a volume fraction of 40%; flow rate: 1 mL/min; elution mode: gradient elution (0 min to 16 min, 100% A to 40% A; 16 min to 20 min, 40% A to 0% A; 20 min to 25 min, 0% A to 100% A); multi-wavelength detection (324 nm for vitamin A; 263 nm for vitamin D; 290 nm for vitamin E; 254 nm for vitamin K; and 450 nm for carotenoid); column temperature: 25° C., and injection volume: 10 μL.

(82) This quantitative analysis method is accurate and reliable. The sample recovery rate of the detected fat-soluble vitamins and carotenoids is 87.0% to 112.5%, and the minimum detection limit is 0.005 ug/mL to 0.025 ug/mL. As detected by the experiment, the contents of vitamin A, 25-hydroxyvitamin D.sub.3, α-tocopherol, γ-tocopherol, lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene and lycopene in the serum of 20 normal people are 3.05±0.93 μmol/L, 0.081±0.026 μmol/L, 54.2±6.5 μmol/L, 5.69±3.13 μmol/L, 0.23±0.14 μmol/L, 0.055±0.024 μmol/L, 0.19±0.04 μmol/L, 0.062±0.043 μmol/L, 0.72±0.32 μmol/L, and 0.29±0.06 μmol/L, respectively, which are consistent with the content ranges of these nutrients in a normal person reported in references.

Example 11

(83) A method for simultaneously determining fat-soluble vitamins and carotenoids in serum included the following steps:

(84) 1) 5 uL of a solution of retinyl acetate and trans-β-apo-8′-carotenal in methanol with a known concentration was added to 250 uL of serum, and the resulting mixture was well mixed;

(85) 2) then 100 uL of a solution of N-dodecylpyridinium tetrafluoroborate ([C12Py][BF.sub.4]) in n-hexane with a mole fraction of 20% was added to the serum, the resulting mixture was vortexed for 2 min and then centrifuged in a 4° C. centrifuge at 12,000 r/min for 10 min, and the L extract phase was collected;

(86) 3) a certain volume of each of stock solutions for fat-soluble vitamin and carotenoid standards was taken and then diluted with simulated serum to give standard solutions with a series of concentrations, and 250 uL of each of the standard solutions was taken and treated according to steps 1) and 2) to give the L extract phases of the standards; and

(87) 4) the serum extract phase and the standard extract phases were analyzed by LC, the internal standard method was adopted to establish standard curves for fat-soluble vitamins and carotenoids, and the peak area ratio of each to-be-tested component in serum to the internal standard was compared with the standard curve to obtain the content of each fat-soluble nutrient in serum.

(88) The HPLC analysis was conducted at the following conditions: chromatographic column: YMC C30; mobile phase A: a methanol aqueous solution with a volume fraction of 95%; mobile phase B: a mixed solution of i-propanol/n-hexane with a volume fraction of 75%; flow rate: 1 mL/min; elution mode: gradient elution (0 min to 20 min, 80% A to 0% A; 20 min to 23 min, 0% A to 0% A; 23 min to 30 min, 0% A to 100% A); multi-wavelength detection (324 nm for vitamin A; 263 nm for vitamin D; 290 nm for vitamin E; 254 nm for vitamin K; and 450 nm for carotenoid); column temperature: 25° C., and injection volume: 10 μL.

(89) This quantitative analysis method is accurate and reliable. The sample recovery rate of the detected fat-soluble vitamins and carotenoids is 93.7% to 106.4%, and the minimum detection limit is 0.005 ug/mL to 0.025 ug/mL. As detected by the experiment, the contents of vitamin A, 25-hydroxyvitamin D.sub.3, α-tocopherol, γ-tocopherol, lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene and lycopene in the serum of 30 normal people are 3.18±0.23 μmol/L, 0.087±0.034 μmol/L, 43.2±15.2 μmol/L, 8.14±1.90 μmol/L, 0.38±0.26 μmol/L, 0.065±0.029 μmol/L, 0.21±0.05 μmol/L, 0.071±0.037 μmol/L, 0.83±0.23 μmol/L, and 0.29±0.07 μmol/L, respectively, which are consistent with the content ranges of these nutrients in a normal person reported in references.

(90) The above contents are merely used to illustrate the technical ideas of the disclosure, rather than to limit the protection scope of the disclosure. Any variations made based on the technical solutions according to the technical ideas proposed by the disclosure shall fall within the protection scope as defined by the claims of the disclosure.