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User Benefits Application News Experimental Reagents and standards Retinol, pyrogallol, potassium hydroxide, and butylated hydroxytoluene were purchased from Sigma-Aldrich. A fat-soluble Vitamin Kit (containing Vitamin D3, Vitamin E and Vitamin K1) was purchased from AccuStandard. Hexane and reagent alcohol of HPLC grade were used. Formic acid of LCMS grade were used as additives in the mobile phase prepared from LC/MS grade Methanol and Milli-Q water. LC-MS/MS conditions The analytical conditions on LCMS-8045 are compiled in Table 1. Quantitative Determination of Fat-Soluble Vitamins in Infant Formula by LC-MS/MS Method with Supported Liquid Extraction (SLE) A LC-MS/MS method with sample preparation using supported liquid extraction (SLE) column is established for quantitative determination of fat-soluble vitamins (A, 1, D3, E and K1) in infant formula on LCMS-8045. The reliability of the method was verified and confirmed with analysis of fat-soluble vitamins in FAPAS QC quality control material. The analysis results indicate the acceptable accuracy and precision of the vitamin analysis. Lai Kit Yee 1 , Xin Jie 1 , Zhaoqi Zhan 1 1 Shimadzu Asia Pacific, Singapore Fat-soluble vitamins / LCMS-8045 Introduction Fat-soluble vitamins are classified into four categories: Vitamin A, D, E and K. They have important roles in several functions of the human body, such as vision (vitamin A), calcium absorption (Vitamin D), antioxidative protection in cell membranes (Vitamin E), and blood coagulation (Vitamin K). Infant formula, as a main source of feeding for infants, must be able to provide sufficient vitamins. Due to complexity and instability of Fat-soluble vitamins during extraction, their quantitation remains challenging. Thus, extraction is the critical step in determination of fat-soluble vitamins in milk samples. [1] In this application notes, a sensitive MRM method is described for the quantitative determination of fat- soluble vitamins in infant formula with supported liquid extraction (SLE) on LCMS-8045, a tandem LC-MS/MS system with an APCI interface. *FAPAS: “Food analysis performance Assessment Scheme” is the proficiency scheme for food and water testing, organized by the Food and Environment Research Agency, UK. Table 1 Analytical conditions on LCMS-8045 MS Conditions (LCMS-8045) Interface APCI Interface Temp. 350 o C DL Temp. 250 o C Heat Block Temp. 200 o C Nebulizing Gas 3 L/min Drying Gas Flow 5 L/min Mode MRM, Positive mode Sample preparation A schematic procedure of sample preparation is shown in Fig. 1. Quality control material were prepared according to the procedure. Name Quantifier CE (V) Ref. Ion CE (V) Vitamin A1 269.3> 92.9 -24 269.3>107.2 -18 Vitamin D3 385.4>259.1 -14 385.4>367.2 -11 Vitamin E 431.5>165.2 -21 431.5>137.3 -43 Vitamin K1 451.5>187.1 -25 451.5>57.2 -35 Table 2 MRM transitions and optimized CE LC Conditions (Nexera) Column Flow Rate Mobile Phase LC program Oven Temp. Injection Vol. Elution gradient Shim-pack TM GIST C18 column (2.1 x 100 mm, 3 µm) P/N: 227-30008-05 0.4 mL/min A: 0.1% Formic acid in Mili-Q water B: 0.1% Formic acid in Methanol Gradient elution, 15 minutes 40 o C 10 µL B%: 80% (0.5min)→ 100% (5min to 12 min)→ 80% (12.01min to 15min) The MRM transitions (quantifier ion and reference ion) and their optimized collision energy (CE) of the four compounds are compiled into Table 2.
Application News Table 3 Linearity, LOD, LOQ and %RSD of fat-soluble vitamins Calibration curve and sensitivity The calibration curves were established with mixed standards in pure solvent from 10 ng/mL to 400 ng/mL (Fig. 3). Good linearity was obtained for all vitamins with R 2 value greater than 0.999. The repeatability was evaluated by 6 consecutive injections of mixed standards solution at 50ng/mL concentration. The linearity, LOD, LOQ and %RSD results are tabulated in Table 3. Fig. 1 Sample preparation procedure of infant formula sample by supported liquid extraction (SLE). Fig. 2 MRM peaks of 50 ng/mL mixed standards Fig. 3 MRM calibration curves of vitamin A1, D3, E, and K1 in the range of 10~600 ng/mL . Determination of Quality Control Material (QCM) The established method was applied to analyze FAPAS quality control material (T21125QC) [3] to determine the reliability of the method. Six separate analyses were performed on the QC material. The analysis results of the QC material were summarized in Table 4. %RSD of the 6 results are less than 10%, indicating acceptable precision of the method. Table 4 Results of 6 analysis on QC material T21125QC. 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 min 0 5.0e4 1.0e5 1.5e5 2.0e5 Vitamin A1 Vitamin D3 Vitamin E Vitamin K1 Saponification • Add 1.5mL of Mili-Q water to 0.1g of milk powder and sonicate. • Add 2mL 2% pyrogallol in reagent alcohol and 1mL of 50% KOH. • Mix overnight. pH Adjustment (target pH around 6-7) • Add 250μL Formic acid and mix. • Adjust pH of the sample solution to around 6-7. Extraction • Load all sample solution into SLE column, wait for 5mins • Add 8mL of 0.1% BHT in hexane and allow to flow under gravity. • Add 2 x 8mL of hexane • Collect the eluent in a glass tube. • Apply vacuum for 10-30 seconds to complete elution. • Blow dry eluent under gentle stream of Nitrogen. Reconstitution and Filtration • Reconstitute with 0.5mL of Methanol. • Filter with 0.22μm Nylon membrane filter into a 1.5mL amber vial. • Analyse with LCMSMS. Vitamin RT (min) R 2 LOD (ng/mL) LOQ (ng/mL) %RSD Area (n=6) %RSD RT (n=6) A 4.090 0.9998 2.8 8.5 0.44 0.22 D3 6.499 0.9997 2.1 6.3 3.41 0.06 E 7.025 0.9998 1.6 4.8 0.73 0.03 K1 8.561 0.9998 2.5 7.4 2.05 0.02 Quantitation results of all six analyses are within the acceptable range of the QC material. The average concentration of the six analysis, assigned value and their acceptable range are shown in Table 5. Sample A (μg/100g) D3 (μg/100g) E (μg/100g) K1 (μg/100g) QC1 436.8 10.2 22.1 31.4 QC2 450.0 9.7 22.1 33.0 QC3 444.1 9.1 21.3 39.4 QC4 436.7 8.9 21.6 36.0 QC5 468.3 9.3 22.6 37.4 QC6 455.7 8.7 21.9 36.0 %RSD 2.3 4.8 1.7 6.9 Conc. (ppb) 0 200 400 600 Area 0.0e0 5.0e5 1.0e6 1.5e6 2.0e6 Vitamin A1 y = 3377.900x - 9347.817 R² = 0.9998011 R = 0.9999005 Curve Fit: Default (Linear) Weighting: Default (None) Zero: Default (Not Forced) Conc. (ppb) 0 200 400 Area 0.0e0 2.0e5 4.0e5 6.0e5 8.0e5 1.0e6 1.2e6 1.4e6 Vitamin D3 y = 3195.853x - 8296.351 R² = 0.9997755 R = 0.9998877 Curve Fit: Default (Linear) Weighting: Default (None) Zero: Default (Not Forced) Conc. (ppb) 0 200 400 Area 0.0e0 1.0e6 2.0e6 3.0e6 4.0e6 5.0e6 6.0e6 Vitamin E y = 11110.17x - 81655.83 R² = 0.9998990 R = 0.9999495 Curve Fit: Default (Linear) Weighting: Default (None) Zero: Default (Not Forced) Conc. (ppb) 0 200 400 Area 0.0e0 1.0e6 2.0e6 3.0e6 4.0e6 5.0e6 6.0e6 Vitamin K1 y = 11313.97x - 45715.51 R² = 0.9998969 R = 0.9999484 Curve Fit: Default (Linear) Weighting: Default (None) Zero: Default (Not Forced) Results and Discussion Optimization of LC-MS/MS MRM optimization of 4 non-polar fat-soluble vitamins were performed in APCI positive mode. To separate the fat-soluble vitamins, a C18 column was used. Initial percentage of mobile phase B was set at 80% to shorten the total analysis time and to achieve sharper peaks. Fig. 2 shows the MRM chromatograms of a 50 ng/mL mixed standards in pure solvent. pH adjustment before loading of sample onto the SLE column is important as it will affect the retention of fat- soluble vitamins in the column. Experiment result shows that sample pH of around 6-7 would be the best condition for good recovery of fat-soluble vitamins.
Application News Copyright © 2022 SHIMADZU group. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. This publication may contain references to products that are not available in your country. Please contact us to check the availability of these products in your country. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. See http://www.shimadzu.com/about/trademarks/index.html for details. Third party trademarks and trade names may be used in this publication to refer to either the entities or their products/services, whether or not they are used with trademark symbol “TM” or “”. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. First Edition: April 2022 04-AD-0265-EN Shimadzu Corporation www.shimadzu.com/an/ SHIMADZU (Asia Pacific) Pte. Ltd, www.shimadzu.com.sg Fig. 4 MRM peaks of vitamin A, D3, E and K in QC material. References 1. E. Karrar, Isam A.Mohamed Ahmed, M. F. Manzoor,W.W., F.Sarpong, X.Wang, Food Chemistry 373 (2022) 131436 2. Y.L Chew, Shimadzu Appl. News AD-0208 3. FAPAS QC Material Data Sheet (T21125QC) Conclusion An LC-MS/MS method was established for the quantitative determination of fat-soluble vitamins including vitamin A1, D3, E and K1 in infant formula. Sample preparation using a supported liquid extraction (SLE) column was adopted. Good linearity was achieved with R 2 > 0.999 in a range of 10-400 ng/mL. Analysis of a QC material (FAPAS) verified that the method is well acceptable in accuracy and precision without use of internal standards. Acknowledgement We would like to thank Teh Bo Yan who participated in this work during his internship at Shimadzu. Table 6 Inter-day %RSD of RT and concentration of vitamin A, D3, E and K1 detected in QC material T21125QC (n=6 x 2 days). Inter-day repeatability of the method was evaluated. %RSD of retention time and concentration are tabulated in Table 6. Inter-day repeatability were found to be less than 10% for vitamin A1 and vitamin E, and less than 15% for vitamin D3 and vitamin K1. Vitamin Ave. Conc., μg/100g %RSD, RT %RSD, Conc. A 437.9 0.33 3.5 D3 10.0 0.09 10.8 E 21.8 0.09 3.6 K1 38.0 0.11 14.9 6.97e5 Q 269.25>92.90 (+) RT=3.939 RT=4.069 RT=4.326 3.5 4.0 4.5 0.0e0 2.0e5 4.0e5 6.0e5 Vitamin A1 Vitamin D3 5.55e5 Q 431.45>165.15 (+) RT=7.018 6.5 7.0 7.5 0.0e0 2.0e5 4.0e5 Vitamin E 9.76e4 Q 451.45>187.10 (+) RT=8.170 RT=8.564 8.0 8.5 9.0 0.0e0 5.0e4 Vitamin K1 2.80e4 Q 385.40>259.10 (+) A=56781 H=14264 RT=6.492 C=20.3631 RT (min) 5.5 6.0 6.5 7.0 0.0e0 5.0e3 1.0e4 1.5e4 2.0e4 Vitamin Ave. conc. Meas. (n=6) Assigned value %Recovery Acceptable range A 448.6 443 101.3 355-532 D3 9.3 10.6 87.8 7.7-13.5 E 22.0 21.6 101.8 17.3-25.9 K1 35.5 40.6 87.4 25.7-55.5 Table 5 Quantitation results, assigned value and acceptable range of vitamin A, D3, E and K1 in QC material T21125QC (Unit: ug/100 g) Nexera and Shim-pack are trademarks of Shimadzu Corporation or its affiliated companies in Japan and/or other countries.
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