Determination of Vitamin B2 Content in Multivitamin Tablets
Also known as Riboflavin, Vitamin B2, or B2, is one of the 13 vitamins essential to the function of the human body. An important component of many of the body’s enzyme systems, it is involved in both material and energy metabolism. Primarily, it serves to promote metabolism as a coenzyme, and it is an essential factor in the growth of humans and many animals.
Aside from occurring naturally in a number of foods, and being added to others as a form of food fortification, it is also found being sold as a dietary supplement. These are often just the Vitamin, but can also take the form of multivitamin tablets.
This report describes the method by which of the L-3000 HPLC System can be used to accurately and reliably determine the Vitamin B2 content of multivitamin pills. Such a determination, and it’s accuracy, would be of interest to not only to a producer for quality control purposes, but also in adhering to countries’ regulatory parameters.
Vitamin B2 Content Assay
The following equipment were used in the determination of aspirin content in enteric-coated aspirin tablets experiment: A 4-piece Dionamix HPLC system (Consisting of an L-3100 Solvent Organizer, an L-3220 Binary High-Pressure Gradient Pump, an L-3300 Autosampler, and an L-3500 UV/Visible Detector), a 5 µm, 4.6mmx250mm Dionamix Compass C18, a Millipore Milli-Q ultrapure water purification system, a Sartorius Germany BT 124S scale, a set of Eppendorf 10-100µL and 10-1000µL pipettes, and a Tianjin Autoscience Ultrasound.
The experiment ran for 30 minutes, and was carried out under the following parameters: A flow rate of 1.0mL/min, an injection volume of 10µL, a detection wavelength of 444nm, and a column temperature of 30°C.
The mobile phase was prepared from a mix of 0.025mol/L sodium heptane sulfonate in 0.5% glacial acetic acid and acetonitrile, at a ratio of 80:20.
Standard Curve Solutions
12.5mg of vitamin B2 was added to a 25mL volumetric flask and mixed with a 0.25% aqueous glacial acetic acid solution to produce a solution with a concentration of 500µg/mL (Stock Solution). 700µL of that solution was extracted and added to a 10mL volumetric flask, and mixed with a 0.25% aqueous glacial acetic acid solution to produce a solution with a concentration of 35µg/mL (Standard Solution (1)). The process was repeated 4 more times, extracting 500µL, 400µL, 300µL, and 100µL of the stock solution to produce solutions with concentrations of 25µg/mL (2), 20µg/mL (3), 15µg/mL (4), 5µg/mL (5).
Standard Curve Solutions
The coating of 1.50g of multivitamin tablets was removed, and the tablets ground down. After grinding, 0.500g was added to a 25mL Erlenmeyer flask with a stopper. 20mL of 0.25% acetic acid water was added, and the mixture was then sonicated for 10 minutes. After, 0.25% acetic acid water was added to the mark and shaken, and then filtered with a 0.45µm membrane (Sample Solution.) 2mL of Standard Solution (3) was mixed with 0.500g of the ground down powder and processed by the same method as above to obtain a 'marked' sample with an additional 80µg/mL.
Standard Solution (3) was injected. It was found to have a retention time of 13.500 seconds and a tailing factor of 0.977 with a column efficiency of 14936. The chromatogram of the aspirin reference substance can be seen on the right.
System Suitability Repeatability
Standard Solution (3) was injected 6 times. The qualitative and quantitative chromatogram of the vitamin B2 reference substance can be seen on the left. A qualitative RSD% (n=6) of 0.144 and a quantitative RSD% (n=6) of 0.577 were calculated from the values below. In the original, the names were a single cell labelled 'Vitamin B2,' but this editor doesn't allow me to merge cells like that.
|Name||Retention Time||Peak Area|
Linear Range of the Standard Curve
Standard Solutions (1), (2), (3), (4), and (5) were successively injected once each, the chromatogram of which can be seen on the right
The standard curve of the vitamin B2 reference substance can be seen on the left. The concentration range was found to be 5µg/mL - 35µg/mL, the regression equation Y = 13.70268 * X, and the correlation coefficient r = 0.9999
Quantification and Detection Limit
Single-point external standard was used to calculate the LOQ and LOD with the lowest concentration in the standard curve. Noise was cancelled via the ASTM method. The chromatogram of Standard Solution (5) can be seen on the right. With a concentration of 5µg/L, a peak height mAU of 3.358, a noise of mAU 0.0357, the LOD (mg/L) S/N = 3 was calculated to be 0.1595 and the LOQ (mg/L) S/N = 10 to be 0.532.
Sample Measurement Results
The multivitamin sample solution was injected, the chromatogram of which can be seen on the left. With a detection concentration of 21.864, a 1.50g sample size, a column efficiency of 8321 and degree of separation of 10.991, the ingredient content was calculated to be 1.640mg/tablet, using the content calculation formula below.
Vitamin B2 Content (mg/tablet) =
AS * 25 * (1.5 / 0.5) / 1000
Where AS is the detected concentration of vitamin B2 in the multivitamin tablet solution (µg/mL)
Sample Measurement Repeatability
The multivitamin sample solution was injected was injected 6 times. The qualitative and quantitative chromatogram of the vitamin B2 sample solution can be seen on the right. A qualitative repeatability RSD% (n=6) 0.310 and a quantitative repeatability of RSD% (n=6) of 0.412 were calculated from the values below. In the original, the names were a single cell labelled 'Multivitamin Tablet,' but this editor doesn't allow me to merge cells like that
|Name||Retention Time||Peak Area|
Rate of Recovery
The 'marked' sample solution was injected 3 times, the chromatogram of which can be seen on the left. Recovery rates were calculated from the formula below.
Rate of Recovery (%) =
(AR - AS) / CA * 100%
AR is measured concentration of the ‘marked’ sample (µg/mL),
AS is the 6-pin average of the measured concentration of the sample (µg/mL), and
CA is the concentration after adding the standard after pretreatment (µg/mL)
Of the three injections, the AR were given at 24.360, 24.382, and 24.378. All three shared an AS of 21.12, and a CA of 3.2µg/mL, giving the calculated rate of recoveries as 101.25%, 101.94%, and 101.81%, with an average rate of recovery of 101.67%
The L-3000 HPLC is capable of quickly and efficiently carrying out the vitamin B2 in multivitamin tablet content experiment to a high degree of accuracy, and with good qualitative and quantitative repeatability.
This makes the Dionamix L-3000 HPLC system and method well suited to the needs of research scientists, quality control managers, industry regulators, and other similar roles that require a precise understanding of the content found within dietary supplement tablets or similar.
Applicable to research and quality assurance processes, the HPLC system and method also delivers more accurate results than other methods, and is additionally quite versatile.