Test Kit for Testing Fat-soluble Vitamins in Serum Using High Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry

Abstract

The present invention describes a test kit for the detection of fat-soluble vitamins in serum using a method based on high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The internal standard (IS) solution included in the kit is based on methanol, acetonitrile and isopropyl alcohol solvents. Ammonium acetate is added to the IS solution to make it more stable and last for longer storage time. The application of this kit can significantly improve the recovery and detection sensitivity of vitamins A, E, K1 and K2 in serum without additional sample enrichment, make sample preparation process simpler and more efficient, keep processed samples stable for longer time, and lower the overall cost for more accurate and repeatable test results.

Claims

1. A test kit based on HPLC-MS/MS for detecting fat-soluble vitamin in serum comprising: an internal standard solution, wherein the internal standard solution comprises stable isotope labeled fat-soluble vitamin and an organic solvent for denaturing protein in the serum, and wherein the solvent includes methanol, acetonitrile and isopropanol.

2. The test kit according to claim 1, wherein the internal standard solution comprises a additive, and wherein the additive is selected from the group of formic acid, ammonium fluoride, ammonium formate, ammonium acetate, or 2,6-di-tert-butyl-4-methylphenol.

3. The test kit according to claim 2, wherein the additive is ammonium acetate.

4. The test kit according to claim 3, wherein the concentration of ammonium acetate is 10˜50 mM.

5. The test kit according to claim 4, wherein the volume ratio of methanol to acetonitrile to isopropanol is 5˜75%:5˜75%:5˜75%.

6. The test kit according to claim 5, wherein the volume ratio of methanol to acetonitrile to isopropanol is 70˜75%:15˜25%:5˜10%, and wherein the concentration of ammonium acetate is 40˜50 mM.

7. The test kit according to claim 5, wherein the test kit further comprises a calibration solution, and the calibration solution includes one or more of vitamin A, 25-hydroxy vitamin D2 (25-OH vitamin D2), 25-hydroxy vitamin D3 (25-OH vitamin D3), vitamin E, vitamin K1 and vitamin K2.

8. The test kit according to claim 7, wherein the calibration solution matrix is human serum.

9. The test kit according to claim 8, wherein the test kit further comprises three quality control solutions and wherein each of the quality control solution includes serum matrix sample with three different concentrations of fat-soluble vitamins: low, medium and high.

10. The test kit according to claim 1, wherein the test kit further comprises two type of mobile phase solutions for liquid chromatography: mobile phase solution A and B; and herein the mobile phase solution A is 0.1% formic acid in water and B is 0.1% formic acid in methanol.

11. The test kit according to claim 1, wherein the fat-soluble vitamin in serum is selected from the group consisting of vitamin A, 25-OH vitamin D2, 25-OH vitamin D3, vitamin E, vitamin K1 or vitamin K2.

12. The test kit according to claim 11, wherein the fat-soluble vitamin in serum is vitamin K2 or vitamin K1.

13. The test kit according to claim 1, wherein the isotope labeled fat-soluble vitamin is one of more of VA-d6, 25-OH VD3-d6, 25-OH VD2-d6, VK1-d4 and VE-d6.

14. A protein denaturing agent for extracting a fat-soluble vitamins from serum comprising: methanol, acetonitrile and isopropanol.

15. The protein denaturing agent according to claim 14, wherein the protein denaturing agent further comprises a additive which is selected from the group consisting of formic acid, ammonium fluoride, ammonium formate, ammonium acetate, and 2,6-di-tert-butyl-4-methylphenol.

16. The protein denaturing agent according to claim 15, wherein the additive is ammonium acetate.

17. The protein denaturing agent according to claim 16, wherein the concentration of ammonium acetate is 10-50 mM.

18. The protein denaturing agent according to claim 16, wherein the volume ratio of methanol to acetonitrile to isopropanol is 5˜75%:5˜75%:5˜75%.

19. The protein denaturing agent according to claim 17, wherein the volume ratio of methanol to acetonitrile to isopropanol is 70˜75%:15˜25%:5˜10%, and the concentration of the ammonium acetate is 40˜50 mM.

20. The protein denaturing agent according to claim 17 wherein the fat-soluble vitamin in serum is one or more of vitamin A, 25-OH vitamin D2, 25-OH vitamin D3, vitamin E, vitamin K1 and vitamin K2.

Description

DESCRIPTION OF THE FIGURE

[0059] FIGS. 1A-1E are the chromatographs of the six fat-soluble vitamins analyzed in embodiment 1. The concentration of each vitamin is the lowest concentration (S1) of each serially diluted vitamin standard dissolved in blank human serum.

EXAMPLES

[0060] The present invention will be further described in detail below with reference to the accompanying figures and embodiments or samples. It should be pointed out that the embodiments described below are intended to facilitate the understanding of the present invention and do not have any limiting on the claims. The reagents used in the following embodiments are all commercially available products that can be purchased.

Embodiment #1: Sample Preparation, Detection and Data Analysis

[0061] Sample Preparation

[0062] 1. Preparation of Standard Solutions and Quality Control Solutions

[0063] Vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E, vitamin K1, and vitamin K2 standard solutions are mixed to make a stock solution. Standard solution and quality control solution are diluted from the stock solutions and mixed with blank human serum (without fat-soluble vitamins) or human serum at a volume ratio of 1:49, respectively.

[0064] Each of the six fat-soluble vitamins has 10 serially diluted concentrations in the standard solution (S1˜S10), which are specified in table 1.

TABLE-US-00004 TABLE 1 10 serially diluted concentrations (S1~S10) of the six fat soluble vitamin in standard curve 25-OH 25-OH Vitamin Vitamin Vitamin Vitamin Vitamin Vitamin ng/ml A D2 D3 E K1 K2 S1 40 2 3 500 0.10 0.10 S2 60 3 4.5 750 0.15 0.15 S3 100 5 7.5 1250 0.25 0.25 S4 160 8 12 2000 0.40 0.40 S5 200 10 15 2500 0.50 0.50 S6 400 20 30 5000 1.00 1.00 S7 600 30 45 7500 1.50 1.50 S8 1000 50 75 12500 2.50 2.50 S9 1600 80 120 20000 4.00 4.00 S10 2000 100 150 25000 5.00 5.00

[0065] In the quality control solutions, each of the six fat-soluble vitamins has three concentrations: low, medium, and high, which are specified in table 2.

TABLE-US-00005 TABLE 2 Three concentrations of the six fat-soluble vitamins in quality control solutions 25-OH 25-OH Vitamin Vitamin Vitamin Vitamin Vitamin Vitamin ng/ml A D2 D3 E K1 K2 Low 144 7.2 10.8 1800 0.36 0.36 Medium 480 24.0 36.0 6000 1.20 1.20 High 1440 72.0 108.0 18000 3.60 3.60

[0066] 2, Preparation of Internal Standard(IS) Solutions

[0067] (1) Preparation of the Internal Standard Solutions

[0068] The concentrations of the internal standard solutions of Vitamin A-d6, 25-OH VitaminD2-d3, 25-OH VitaminD3-d6, vitamin E-d6, and vitamin K-d4 are 3.0 μg/mL, 1.0 μg/mL, 1.0 μg/mL, 20 μg/mL, and 0.025 μg/mL in methanol, respectively.

[0069] (2) Preparation of the Internal Standard Solutions

[0070] Mix 750 ml methanol, 150 ml acetonitrile, and 100 ml isopropanol to make a 75:15:10methanol/acetonitrile/isopropanol mixture; weigh 3.85 g ammonium acetate and add it to the above mixture to make solution A, which is 50 mM ammonium acetate in 75% methanol, 15% acetonitrile and 10% isopropanol.

[0071] Transfer 2 ml internal standard solution to 248 ml solution A (v/v 1:124).

[0072] Although ammonium acetate is used in this embodiment, it has been demonstrated repeatedly that formic acid, ammonium fluoride, ammonium formate, or 2,6-di-tert-butyl-4-methylphenol can also be used in place of ammonium acetate, which will exert the synergistic effect in elevating the recovery rate of the six fat-soluble vitamins in human serum samples.

[0073] Sample Processing

[0074] Human serum sample, standard solution, and quality control solutions are processed in the same manner as following:

[0075] (1) Transfer 50 μL of each above solution(Human serum sample, standard solution, and quality control solutions) into one well of the wells of a 96-deepwell plate

[0076] (2) Add 250 μL IS solution to the same well and vortex the plate to mix at 1000 rpm for 10 minutes.

[0077] (3) Spin the plate in a centrifuge at 4,000 rpm for 10 minutes, transfer 200 μL of the supernatant to a new low adsorption 96-well plate and wait to be tested.

[0078] Sample Analysis

[0079] Inject 40 μL of each processed extract(step (3)) into the HPLC-MS/MS system using the following setting:

[0080] Masspectrometer: AB SCIEX Triple Quad 4500MD; chromatographic column: Phenomenex Luna C18 (3 μm, 50×2.0 mm); mobile phase solution A: 0.1% formic acid in water; mobile phase solution B: 0.1% formic acid in methanol; chromatographic flow rate: 0.7 mL/min; column temperature: 40° C.; injector temperature: 15° C.; injection volume: 40 μL.

[0081] Gradient elution program is detailed in table 3.

TABLE-US-00006 TABLE 3 Gradient elution program time Mobile Mobile Flow rate (min) phase A % phase B % (ml/min) 0.00 60.0 40.0 0.70 4.50 10.0 90.0 0.70 5.00 0.0 100.0 0.70 6.70 0.0 100.0 0.70 6.71 60.0 40.0 0.70 7.00 60.0 40.0 0.70

[0082] The retention times of the six fat-soluble vitamins are: 2.84 minutes for vitamin A, 2.75 minutes for 25-OH vitamin D2, 2.69 minutes for 25-OH vitamin D3, 5.03 minutes for vitamins E and K2, and 5.69 minutes for vitamin K1.

[0083] As illustrated in FIGS. 1A-1E, the six vitamins are separated after liquid chromatography and eluted at different retention times into mass spectrometer, in which each vitamin is detected by selected ion monitoring and its concentration is determined.

[0084] The six fat-soluble vitamins separated through liquid chromatography are eluted into a mass spectrometer for detection. Atmospheric pressure chemical ionization (APCI) and multiple reaction monitoring (MRM) scan mode are used to detect the six fat-soluble vitamins. A standard curve is also made to calculate the concentrations of the six vitamins.

[0085] Segmented data collection in mass spectrometer is as following: the first segment includes vitamin A, 25-OH-vitamin D2, and 25-OH-vitamin D3; the second segment includes vitamin E, vitamin K1, and vitamin K2.

[0086] The parameters of the mass spectrometer are detailed in table 4.

TABLE-US-00007 TABLE 4 Parameters of the mass spectrometer Ionization mode APCI (+) Scan Mode MRM Spray voltage +4500 V temperature 450° C. Sheath gas 50 L/min Auxiliary gas 50 L/min Curtain gas 30 L/min data collection first: 0-3.5 min segment second: 3.5-7 min

[0087] The m/z of parent/product ion pair, declustering potential (DP), collision energy (CE), and collision cell exit potential CXP) for detecting each vitamin is listed in Table 5.

TABLE-US-00008 TABLE 5 Parent/product pair m/z and other parameters Analyte and internal standard Q1 m/z Q3 m/z DP CE CXP Vitamin A 269.2 119.1 59 25 11 269.2 93.0 66 34 11 25-OH vitamin D2 395.3 271.1 70 19 11 413.3 271.1 70 19 11 25-OH vitamin D3 383.3 257.2 103 21 11 401.3 257.2 68 22 11 Vitamin E 431.4 137.1 80 60 11 431.4 109.0 83 61 11 Vitamin K1 451.3 187.1 92 34 11 451.3 225.1 73 27 11 Vitamin K2 445.2 187.2 75 29 11 445.2 81.1 75 29 11 Vitamin A-d6 275.3 122.1 51 30 11 275.3 96.0 68 32 11 25-OH vitaminD2-d3 398.3 274.1 70 16 11 416.3 274.1 69 16 11 25-OH vitaminD3-d6 389.3 263.2 72 21 11 407.3 263.2 43 21 11 Vitamin E-d6 437.4 143.1 66 62 11 437.4 115.1 77 70 11 Vitamin K-d4 455.4 191.1 92 31 11 455.4 229.1 97 27 11

[0088] The fat-soluble vitamins are detected by selecting corresponding ion pairs in selected reaction monitoring at specified retention time, and quantified by using corresponding internal standards.

[0089] After being separated by liquid chromatography, different fat-soluble vitamins are eluted at different retention times, detected and quantified using multiple reaction monitoring mode of the mass spectrometer. Serially diluted internal standard solutions are mixed with reference blank human serum and analyzed the same as for samples. FIGS. 1A-1E demonstrate a typical chromatography of detection of the six fat-soluble vitamins, which shows that using the method described in the present invention can detect all six fat-soluble vitamins simultaneously and accurately.

[0090] Data Processing and Analysis

[0091] 1. Making Standard Curve

[0092] The standard curve is made by plotting the ratio of a target vitamin's peak area to its internal standard's peak area on the y-axis against the known concentration of the internal standard on the x-axis for linear regression analysis. The linear regression equation and correlation coefficient (r) are listed in Table 6.

TABLE-US-00009 TABLE 6 Standard curve linear regression equations and correlation coefficients Analyte linear regression equations and correlation coefficients Vitamin A y = 0.05055*x + 0.09145 (r = 0.99845) 25-OH vitamin D2 y = 0.04916*x + 0.1723 (r = 0.99906) 25-OH vitamin D3 y = 0.05218*x + 0.27584 (r = 0.99920) Vitamin E y = 0.04822*x + 0.13845 ( r = 0.99898) Vitamin K1 y = 0.05207*x + 0.19085 (r = 0.99553) Vitamin K2 y = 0.05064*x + 0.2871 (r = 0.99721)

[0093] 2. Accuracy, Precision and Matrix Effect

[0094] (1) Accuracy and Precision

[0095] The ratios of a target vitamin's peak area to its internal standard's peak area in low, medium and high concentration quality control solutions are determined. The ratios are input into the standard curve equation to calculate each vitamin's concentration in the quality control samples. The results from three repeated analyses are used to calculate accuracy and precision. The detected values are acceptable in accuracy when the average values of the three repeats are 85.0% to 115.0% of the actual values and covariance (CV) is ≤15%. Precision is acceptable when the tested values are within 91.2% to 110.5% in three repeated assays of the quality control solutions at all three concentrations. The CV value of 1.46% to 9.25% between different batches of test is accepted.

[0096] (2) Matrix Effect

[0097] The matrix effect is determined by comparing the concentration of a pure standard solution to the detected value when it is mixed with a blank matrix. When the detected value in a matrix is 85% to 115% of the value of the pure standard, the matrix effect is negligible. When a matrix effect is present, it should be consistent among the quality control samples at all three concentrations. It is acceptable when matrix factor normalized by internal standard is between 95%-106% and CV is between 1.75%˜8.76%.

[0098] 3. Calculation of the Concentrations of the Six Fat-Soluble Vitamins in Human Serum Samples

[0099] The ratio of each vitamin's peak area to its internal standard's peak area in LC-MS/MS is calculated first, and the concentration of each vitamin in a serum sample is then calculated by input this ratio into the corresponding standard curve equation.

Embodiment 2: Stability Test of Internal Standard Solutions in Different Compositions Under Long Term Storage

[0100] In this embodiment, internal standard solutions containing the isotope-labeled fat-soluble vitamins are prepared according to the method described in embodiment 1. The compositions of the tested internal standard solutions are listed in Table 7. Stability is evaluated by incubating internal standard solutions in 48° C. for six months and observing for turbidity and other abnormalities. After six months, the stored internal standard solution is tested and the result is compared to that from an identically but freshly prepared internal standard solution. The concentration of the internal standard solution used in the stability test is the lowest concentration point in the standard curve (S1). Since all six fat-soluble vitamins demonstrate similar test results, herein only the results form vitamin K1 is presented (Table 7).

TABLE-US-00010 TABLE 7 Stability test of internal standard solution in different solvent systems Deviation of Deviation vitamin K1-d4 of vitamin K1 Shelf solvent observation concentration recovery life 1 75% methanol/ No −0.6% −1.1% ≥2 15% acetonitrile/ abnormality years 10% isopropanol with 50 mM ammonium acetate 2 80% methanol/ No −37.2% −7.3% 3 20% acetonitrile abnormality months with 50 mM ammonium acetate 3 75% methanol/ No −13.9% −9.6% 3 15% acetonitrile/ abnormality months 10% isopropanol 4 80% methanol/ No −65.2% −10.5% 1 20% acetonitrile abnormality week 5 methanol No −91.9% −18.7% 1 day abnormality (freshly made) 6 acetonitrile No −85.8% −20.4% 1 day abnormality (freshly made)

[0101] The results in Table 7 demonstrate that the prepared internal standard solution is the most stable if it consists of ammonium acetate, methanol, acetonitrile and isopropanol at the same time. This internal standard solution behaves almost the same after six months storage at 48° C. and its shelf life can be as long as more than two years. When the internal standard solution consists of ammonium acetate, methanol and acetonitrile or methanol, acetonitrile and isopropanol, their stabilities after six months storage at 48° C. decrease significantly and resulting in significant deviation of the test result from the true value. When the internal standard solution contains only one or both of methanol and acetonitrile, its stability is poorer and the standard solution needs to be freshly prepared and used immediately, making sample preparation process cumbersome.

[0102] Thus, the internal standard solution described in the present invention proves to be very stable. By using this internal standard solution, traditionally separated sample extraction and adding internal standard solution steps can be combined into one single step, greatly simplifying sample preparation process. The internal standard solution can be prepared ahead of experiment and available whenever it is needed, which is quite convenient. Overall this is an optimized internal standard solution that greatly simplifies sample preparation process in clinical test of serum fat-soluble vitamins and has the potential to be widely adopted and applied.

Embodiment 3: Comparison of Fat-Soluble Vitamin Test Results Obtained by Using Internal Standard Solutions in Different Compositions

[0103] In this embodiment, internal standard solutions containing isotope-labeled fat-soluble vitamins are prepared according to the method described in embodiment 1. The compositions of the tested internal standard solutions are listed in Table 8. The concentration of the tested internal standard solution is the lowest concentration point in the standard curve (S1). Samples preparation and analysis are carried out using methods described in embodiment 1. The test results of the six fat-soluble vitamins using internal standard solutions in different solvents are listed in Table 8.

TABLE-US-00011 TABLE 8 Test results using internal standard solutes in different solvents 25-OH 25-OH Internal standard Vitamin vitamin vitamin Vitamin Vitamin Vitamin solvents A D2 D3 E K1 K2 75% methanol/ 54672.9 1596.3 3514.9 56793.2 3480.9 6265.6 15% acetonitrile/ 10% isopropanol with 50 mM ammonium acetate 75% methanol/ 51987.8 1357.4 3264.1 50318.7 2769.4 4874.1 15% acetonitrile/ 10% isopropanol with 50 mM formate acetate 75% methanol/ 48758.3 1468.2 3307.8 49719.6 2708.5 4469.0 15% acetonitrile/ 10% isopropanol with 50 mM 2,6-di-tert-butyl- 4-methylphenol 75% methanol/ 42780.4 1649.2 4107.5 40639.1 2058.7 3499.8 15% acetonitrile/ 10% isopropanol 50% acetonitrile/ 39244.8 2072.0 7084.4 35717.0 2054.5 3698.1 50% isopropanol 50% methanol/ 18902.5 982.8 3320.2 13644.0 1252.5 2254.5 50% acetonitrile acetonitrile 21776.5 1477.1 3979.1 39569.7 1172.9 1873.6 methanol 27287.9 1252.2 5603.3 31360.8 292.1 571.1

[0104] Conclusions can be drawn from the results in Table 8 that when using the solvent system recommended in the current invention (75% methanol/15% acetonitrile/10% isopropanol with 50 mM ammonium acetate), chromatographic peak areas of vitamin A, E, K1 and K2 are significantly increased. Although peak areas of 25-OH vitamin D2 and 25-OH vitamin D3 are slightly reduced, the test is friendly to low amount vitamins in human serum, such as vitamin K1 and K2, and is ideal when all six fat-soluble vitamins need to be analyzed at the same time.

[0105] The clinical reference range of vitamin K in human blood is only 0.13-1.39 ng/mL, thus a test method for vitamin K need to have high sample recovery rate and high test sensitivity. When testing all six fat-soluble vitamins at the same time, the test method should be optimized in favor of vitamin K. Although currently some methods for detecting multiple vitamins have been developed and reported, their recovery rate for vitamin K1 and K2 are not satisfactory, resulting in poor test sensitivity for vitamin K1 and K2 and makes it difficult to assess all six fat-soluble vitamins clinically.

[0106] The results in Table 8 also demonstrate that methanol has poor recovery for vitamin K and cannot meet the sensitivity requirement for testing clinical samples. Using the combination of methanol and acetonitrile can improve the recovery of vitamin K significantly. Since the test substances are all fat-soluble vitamins, according to the rule of “likes dissolve likes”, the introduction of isopropanol into the internal standard solution can further increase the recovery of vitamins A, E, K1, and K2. On the same basis, adding ammonium acetate, ammonium formate or 2,6-Di-tert-butyl-4-methylphenol can create significant synergistic effects. The synergistic effect from using ammonium acetate is particularly obvious, therefore the best solvent system for the internal standard solution is 50 mM ammonium acetate in 75% methanol/15% acetonitrile/10% isopropanol.

[0107] The internal standard solution described in this embodiment of the present invention not only simplifies sample preparation, but also significantly improves the recovery rate of vitamins A, E, and K (especially K1 and K2 which have the lowest amount among fat-soluble vitamins in human serum). Improved target recovery also means increased sensitivity and accuracy of the ensuing LC-MS/MS detection result, meeting requirement of each fat-soluble vitamin's clinical reference range. When using the optimized internal stand solution of 50 mM ammonium acetate in 75% methanol/15% acetonitrile/10% isopropanol for sample preparation, the sensitivities for vitamin A, 25-OH vitamin D2, 25-OH vitamin D3, vitamin E, vitamin K1 and vitamin K2 in subsequent LC-MS/MS detection are as low as 0.4 ng/mL, 0.7 ng/mL, 0.3 ng/mL, 3 ng/mL, 10 pg/ml and 6 pg/mL, respectively.

Embodiment 4: Effect of Different Solvent Ratios in Internal Standard Solution on LC-MS/MS Test Results

[0108] In this embodiment, internal standard solutions containing isotope-labeled fat-soluble vitamins are prepared according to the method described in embodiment 1. The internal standard solution contains ammonium, methanol, acetonitrile, and isopropanol. Different ratios of the four in the internal standard solution are tested (Table 9). The concentration of the internal standard is the lowest concentration point in the standard curve (S1). Sample preparation and analysis are carried out using the method described in embodiment 1. The test results of the six fat-soluble vitamins in internal standard solutions with different solvent ratios are listed in Table 9.

TABLE-US-00012 TABLE 9 Test results of the six fat-soluble vitamins in internal standard solutions with different solvent ratios 25-OH 25-OH Internal standard Vitamin vitamin vitamin Vitamin Vitamin Vitamin solution solvent system A D2 D3 E K1 K2 50 mM ammonium in 54672.9 1596.3 3514.9 56793.2 3480.9 6265.6 75% methanol/ 15% acetonitrile/ 10% isopropanol 40 mM ammonium in 53791.6 1557.9 3764.7 55318.7 3269.4 5687.0 75% methanol/ 15% acetonitrile/ 10% isopropanol 20 mM ammonium in 49287.9 1352.2 3003.9 50360.4 2992.1 4713.3 75% methanol/ 15% acetonitrile/ 10% isopropanol 50 mM ammonium in 54180.8 1613.7 3507.2 56639.1 3398.5 5863.8 70% methanol/ 25% acetonitrile/ 5% isopropanol 50 mM ammonium in 40245.7 2149.5 6389.8 36187.7 2134.6 4231.7 15% methanol/ 60% acetonitrile/ 25% isopropanol 50 mM ammonium in 29385.1 1752.6 5517.5 31360.8 2098.1 3060.1 82% methanol/ 15% acetonitrile/ 3% isopropanol 50 mM ammonium in 21902.4 1681.3 3816.4 24057.0 2151.5 2467.4 50% methanol/ 48% acetonitrile/ 2% isopropanol

[0109] The results in Table 9 indicate that when the concentration of ammonium acetate in the internal standard solution is lowered to 20 mM, the recovery rate of fat-soluble vitamins will decrease, so the optimal concentration of ammonium acetate should be 40-50 mM. Similarly, when the percentage of methanol is increased to 80% or lowered to 15%, the recovery rates of fat-soluble vitamins also decrease. Too high or too low percentages of acetonitrile or isopropanol decrease the recovery of fat-soluble vitamins too. Therefore, based on extensive tests it is concluded that the optimal solvent system for internal standards should be 40-50 mM ammonium acetate in 70˜75% methanol/15˜25% acetonitrile/5˜10% isopropanol.

Embodiment 5: Effect of Sample to Internal Standard Volume Ratio on the Test Results

[0110] In this embodiment, internal standard solutions containing the isotope-labeled fat-soluble vitamins are prepared according to the method described in embodiment 1. The internal standards are dissolved in 75% methanol/15% acetonitrile/10% isopropanol with 50 mM ammonium acetate. Internal standard solution is mixed with serum samples in different volume ratios (Table 10). Sample preparation and analysis are performed according to the method described in embodiment 1. The peak areas of the six fat-soluble vitamins in the lowest concentration of the standard curve (S1) are listed in Table 10.

TABLE-US-00013 TABLE 10 Peak areas of vitamins in different sample to internal standard solution ratios (v/v) Sample to internal 25-OH 25-OH standard solution Vitamin vitamin vitamin Vitamin Vitamin Vitamin volume ratio A D2 De E K1 K2 1:1 40672.9 998.9 2011.2 39793.2 2181.0 4013.7 (50 μL sampe to 50 μL internal standard solution) 1:3 51791.6 1357.9 3364.7 52318.7 3269.4 5973.1 (50 μL sampe to 150 μL internal standard solution) 1:5 54672.9 1596.3 3514.9 56793.2 3480.9 6265.7 (50 μL sampe to 250 μL internal standard solution) 1:7 50180.8 1413.7 3307.2 51639.1 3098.5 5864.7 (50 μL sampe to 350 μL internal standard solution) 1:9 39245.7 1049.5 2189.8 43187.7 2034.6 4135.4 (50 μL sampe to 450 μL internal standard solution)

[0111] The results in Table 10 indicate that different volume ratios of the internal standard solution to the sample can greatly affect the recovery rate of the six fat-soluble vitamins from samples. When the volume ratio of the internal standard solution to the sample is 1:1, the proteins in the sample cannot be completely denatured and precipitated, so the extraction efficiency of the fat-soluble vitamins is reduced and the test result is affected by strong matrix effect. When the volume ratio of the sample to the internal standard solution is 1:9, sample is over-diluted, which reduces target response and affects detection sensitivity. Therefore, the volume ratio of the sample to the internal standard solution is preferably 1:3 to 1:7, and optimal at 1:5.

Embodiment 6: Comparison of the Stability of Processed Samples in Different Solvent Systems and Sample Plates

[0112] In this embodiment, the internal standard solutions are prepared according to the method described in embodiment 1. Internal standard solutions of fat-soluble vitamins in different solvent systems are prepared (Table 11). The standard solutions are processed, put in a sample plate, and stored in the sample chamber for 12 hours before sample injection. The detected concentrations of vitamin K1 in the stored samples and in samples which are processed the same but injected immediately are compared. The deviations of the results from stored samples to the results from samples immediately injected are listed in Table 11.

TABLE-US-00014 TABLE 11 Stability of prepared samples in different solvent system and sample plate Sample Sample Sample Sample Solvent system plate 1 2 3 75% methanol/25% acetonitrile regular −87.2% −74.9% −82.4% 75% methanol/25% acetonitrile −46.7% −47.7% −67.5% with 20 mM ammonium acetate 75% methanol/25% acetonitrile −55.9% −57.5% −56.2% with 50 mM ammonium acetate 70% methanol/25% acetonitrile/ −18.3% −9.5% −19.2 5% isopropanol with 50 mM ammonium acetate 75% methanol/25% acetonitrile Low −15.4% −6.3% 9.1% with 50 mM ammonium acetate adsorption 70% methanol/25% acetonitrile/ 0.8% 1.7% −1.6% 5% isopropanol with 50 mM ammonium acetate

[0113] Vitamin K1 is the least polar analyte among the six fat-soluble vitamins and has a strong tendency to be adsorbed. When it is extracted from the serum using the internal standard solution described in the current invention, it will be adsorbed to the surface of the sample plate well, which could affect the sensitivity and accuracy of its detection.

[0114] It can be seen from Table 11 that in the same type of 96-well plate, using a solvent of 70% methanol/25% acetonitrile/5% isopropanol with 50 mM ammonium acetate that has good solubility for vitamin K1 can reduce the risk of vitamin K1 being adsorbed onto the 96-well plate to some extent, and in turn lower the deviation of detected vitamin K1 value from the actual concentration. By comparing different 96-well plates, it was found that using low adsorption 96-well plate to store sample extracts can increase their stabilities significantly and improve the stability of vitamin K1 test results. Therefor it's preferred to choose low-adsorption 96-well plate in the present invention.

[0115] It is to be understood that in the absence of any elements or limitations disclosed herein, illustrations and descriptions in the present invention may be implemented. Thus the illustrations and descriptions disclosed herein are only explanatory rather than limitations, and they are not intended to exclude any equivalents of the features or partial features illustrated and described in the disclosure. Various modifications are possible and fall within the scope of the present invention. Therefore, it is to be understood that although the present invention is disclosed through various embodiments and optional features, modifications and variations of the concepts described herein may be adopted by others skilled in the art, so these modifications and variations also fall within the scope of the present invention defined by the appended claims.

[0116] Articles, patents, patent applications, all other reference documents and information which are electronically available are included herein for reference, in their entirety to certain extent, the same as an individual publication being specifically referenced. The applicant reserves the right to incorporate any or all materials and information from such articles, patents, patent applications, and other reference documents into the current invention.