Determination of Vitamin B5 (Pantothenic Acid) in Infant Dairy Products in Relation to GB 5009.210-2023
Jack Wang
R&D APAC lab, Shanghai, China
Abstract
In this study, high performance liquid chromatography-mass spectrometry (LC-MS/MS) was used to analyze pantothenic acid (vitamin B5) in infant dairy products. The results show that the method is fast and meets the performance criteria of the GB 5009.210-2023 "Determination of pantothenic acid in Food Safety National Standard." It provides an efficiency improvement over the classical microbiology method and adds robustness compared to a HPLC-UV/DAD method.
Overview of Sections:
Chemical structure of calcium-D-pantothenate (vitamin B5)
Introduction
Pantothenic acid, also known as vitamin B5 (VB5), is a water-soluble vitamin with the chemical formula C9H17NO5. Its name, "pantothenic acid", derives from its widespread presence in both plant and animal sources. Its main physiological function is to form coenzyme A and acyl carrier proteins, and through them play a role in metabolism, participating in the metabolism of carbohydrates, lipids, and proteins in the body.1
Since pantothenic acid cannot be synthesized by the human body itself, it needs to be obtained via food intake. Hence, it is added to many foods, such as infant formulas, special medical formula foods, infant cereal supplements, pregnant women, and nursing mothers’ nutritional supplements or health foods. Pantothenic acid naturally exists in the form of type D, which is unstable to acid, alkali, and heat and is mostly used as its calcium salt as a food additive.2-4
GB 5009.210-2016, "Determination of pantothenic acid in Food Safety National Standard," stipulates that the main methods for the determination of pantothenic acid in food are either a microbiological approach or high-performance liquid chromatography (HPLC). The former is a classical method for the determination of pantothenic acid in all kinds of foods, but it is very time-consuming (the complete pre-treatment process takes about 36 h) and may cause inaccurate quantification due to its narrow linear range. High-performance liquid chromatography (HPLC) with UV or diode array detector (DAD) is the other analytical technique mentioned for the determination of pantothenic acid in nutritional supplements, health food, and infant dairy products.
Because dairy products, such as partially hydrolyzed protein or deeply hydrolyzed protein milk powder, contain significant amounts of protein, the determination of pantothenic acid could be challenging because of matrix interferences caused by the proteins. Consequently, the method's separation standards for distinguishing analyte peaks from matrix peaks were frequently unmet, impacting the accuracy of the final quantitative results. Therefore, an LC-MS/MS method was considered that also enabled external calibration.
In the draft of the new version of GB 5009.210-2023, "Determination of pantothenic acid in the National Standard for Food Safety", which was first published in China in 2021, liquid chromatography tandem mass spectrometry (LC-MS/MS) was added as a method for the determination of pantothenic acid in various complex substrates.5 LC-MS/MS provides the required selectivity and sensitivity and can accurately determine the pantothenic acid content in a short time and further improve the accuracy of the method.
To address the high protein content of the samples, zinc acetate and potassium ferricyanide were used as precipitators in this experiment to reduce the matrix interference effects of proteins. The resulting sample solution can then easily be centrifuged and filtered. In addition, the multiple reaction monitoring (MRM) mode used on the MS system eliminates further interferences from small protein molecules with the target analyte, pantothenic acid.
The here described LC-MS method using an Ascentis® Express C18, 2 μm particle column was aligned to and evaluated against criteria described on the draft of the GB 5009.210-2023 standard.
Experimental
The analysis was performed in accordance with the draft GB method using the following procedures:
Reagent Preparation
- Zinc acetate solution (300 g/L): 30.0 g zinc acetate were dissolved in 100 mL water. This solution is stable at room temperature for three months.
- Potassium hexacyanoferrate solution (c = 150 g/L): 15.0 g potassium hexacyanoferrate were dissolved in 100 mL water. This solution is stable at room temperature for three months.
Standard Preparation
- Pantothenic acid standard solution I (c = 500 mg/L): 136 mg D-calcium pantothenate standard were accurately weighed into a 250 mL volumetric bottle, dissolved in water, volume adjusted to scale, and mixed well. Stored at -18 °C and protected from light, this solution can be kept for three months. (Note: pantothenic acid = calcium pantothenate x 0.920).
- Pantothenic acid standard solution II (c = 10.0 mg/L): Pipette 1.0 mL of pantothenic acid standard solution I was pipetted into a 50 mL amber glass volumetric flask and topped to mark with water.
- Pantothenic acid standard solutions 1-8 (c = 1.0, 5.0, 10.0, 50.0, 100, 300, 500, and 1000 μg/L): 2.5, 5.0, 25, 125, 250, 750, 1250, and 2500 μL of pantothenic acid standard solution II were pipetted into six 25 mL volumetric flasks. Each was filled to the mark with water to obtain pantothenic acid standard solutions 1-8 with concentrations of 1.0, 5.0, 10.0, 50.0, 100, 300, 500, and 1000 μg/L, respectively.
Sample Preparation
- Sample: Infant formula milk powder was obtained from a local supermarket at 400 g/barrel. The food label showed the content of pantothenic acid (VB5) in the milk powder to be 20±2 μg/kg. Note: The later experimental determination showed a content of 20.05 μg/kg, therefore, 20.0 μg/kg was used as the background concentration for subsequent experiments and quantitative calculations.
- Extraction: Five grams of sample were accurately weighed into a glass and dissolved in pure water to a total volume of 50 mL. The solution was stirred with a magnetic stirrer for 10 to 30 minutes until the sample was completely dissolved and ready for the next cleanup process.
- Cleanup: 5.0 mL of the above extraction solution was pipetted into a 50 mL centrifuge tube, filled up to 20 mL with water, and swirled for 10 seconds. 0.4 mL each of zinc acetate solution and potassium hexacyanoferrate solution were added, and the solution was diluted to 25 mL with water. The mixture was vortexed for 10 seconds, allowed to settle for 10 to 30 min, then centrifuged at 8000 rpm for 2 min. The final sample was prepared by filtering the resulting supernatant into an HPLC vial using a 0.22 μm nylon filter membrane.
Recovery and precision
Two samples were prepared from each 5.0 g of infant dairy product samples, using 50 and 100 μL of pantothenic acid standard solution II for spiking. The pantothenic acid spike concentration of the resulting samples was 100 and 200 µg/kg, respectively.
Results & Discussion
Infant formula milk powder was extracted by the described sample preparation and quantitated against and external calibration using LC-MS/MS. An assessment regarding calibration linearity, precision, recovery, sensitivity, and a measurement of an actual infant formula milk powder sample was done.
Standard chromatogram
The results for the LC-MS/MS analysis of pantothenic acid standard solution 1 (c = 10.0 μg/L) are shown in Figure 1. Table 2 displays the chromatographic data for Figure 1.
Figure 1.LC-MS/MS chromatogram of pantothenic acid (1) standard solution 1 (c = 10.0 μg/L). MRM transitions: 220.07 → 89.98 (quantitative).
Calibration
The results for the external calibration experiments in the range of 1.0-1000 µg/L are shown in Figure 2 and Table 3. The linearity of the calibration showed an R2 value of 0.9991, meeting the GB 5009.210-2023 criteria of ≥ 0.9980.
Figure 2.Calibration curve obtained by the analysis of pantothenic acid standard solutions 1-8 (c = 1.00，5.00，10.0, 50.0, 100, 300, 500, and 1000 μg/L).
Data Precision and Recovery
The spiked infant formula milk powder sample, labeled with a background value of 20 µg/kg, was spiked with 100 µg/kg of pantothenic acid (Figure 3) for precision assessment. The results shown in Table 4 are the data calculated after the sample background of 20 μg/kg was deducted. The RSD at the test concentration was 1.0% (GB 5009.210-2023 specifies an RSD limit of ≤ 20%).
The results of the recovery determination experiment using the second spiked sample at a spiking level of 200 µg/kg of pantothenic acid in infant formula milk powder are shown in Table 5. The % recovery was 89.5 to 93.5% (GB 5009.210-2023 specifies a required recovery of 80 - 110%).
Figure 3.LC-MS/MS chromatogram of an infant formula milk powder sample spiked at 100 µg/kg with pantothenic acid (1). MRM transition: 220.07 → 89.98 (quantitative).
Sensitivity
The Limit of Detection (LOD) and Limit of Quantification (LOQ) were calculated according to the GB method using baseline noise of a blank sample: LOD (3N/X); LOQ (10N/X). The LC method’s sensitivity was determined with a limit of detection (LOD) of 0.01 µg/L and a limit of quantification (LOQ) of 0.03 µg/L (Table 6), representing sample concentrations of 0.5 µg/kg and 1.5 µg/kg respectively.
Unspiked Sample Measurement
The results for the unspiked infant dairy product sample (c = 20 µg/kg; the value for pantothenic acid on the nutritional label) are shown in Figure 4. The quantification against the here established calibration resulted in a pantothenic acid (VB5) content of 20.05 µg/kg.
Figure 4.LC-MS/MS chromatogram of the unspiked infant formula milk powder sample (c = 20 µg/kg, value on the nutritional label). MRM transition: 220.07 → 89.98 (quantitative).
For this detection, the lowest point of the standard curve was selected for single-point quantitative analysis according to the requirements of the GB method. The content of detected VB5 was determined at 20.05 µg/kg.
Conclusion
Pantothenic acid (vitamin B5) was extracted from infant formula milk powder by dissolving, centrifuging, and filtering the sample. Quantification by LC-MS/MS using an Ascentis® Express C18, 2 μm particle UHPLC column and an external standard calibration method was carried out according to GB 5009.210-2023 method and fulfilled the stated requirements regarding linearity, precision (RSD), and spike recovery.
The sample handling process was shown to be straightforward and greatly decreased sample pretreatment and instrument run time compared to the traditional microbiology sample pretreatment process, which takes about 36 hours to complete. Therefore, this LC-MS/MS method allows to achieve reliable results significantly faster, and by that, improving the efficiency compared to the microbiological approach. At the same time, the MS/MS method’s additional selectivity, which significantly reduced or eliminated interference from matrix peaks, enabled the use of an external calibration offering an advantage over the HPLC-UV or -DAD method.
REFERENCES
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