Determination of Vitamin B6 Content in Vitamin B6 Tablets
As suggested by the name, the primary ingredient of Vitamin B6 tablets is Vitamin B6. These tablets are suited for the treatment of Vitamin B6 deficiencies and isoniazid poisoning, but can also be used to treat the vomiting and seborrheic dermatitis caused by anticancer drugs and radiation sickness during pregnancy. They can be used to treat infant seizures, or be given to pregnant women to prevent infant seizures.
As the primary ingredient in such tablets, and thus the primary factor in the dosage size. As such, accurate determination of the Vitamin B6 content is of vital important to those performing research, or to those involved at the product quality assurance level.
This report describes the method by which of the L-3000 HPLC System can be used to accurately and reliably determine the Vitamin B6 content in Vitamin B6 tablets.
Vitamin B6 Content Assay
The following equipment were used in the determination of IgG content in bovine colostrum experiment: A 5-piece Dionamix HPLC system (Consisting of an L-3120 Solvent Organizer, an L-3220 Binary High-Pressure Gradient Pump, an L-3400 Column Thermostat, an L-3300 Autosampler, and an L-3500 UV/Visible Detector), a 5µm, 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 15 minutes, and was carried out under the following parameters: A flow rate of 1.0mL/min, an injection volume of 3µL, a detection wavelength of 291nm, and a column temperature of 40°C.
The mobile phase was prepared from an A and B component. Component A was composed of 0.04% sodium heptane sulfonate, adjusted to a pH of 3.0 with glacial acetic acid. Component B was Methanol. The two were mixed with a 75:25 A:B ratio.
Standard Curve Solutions
0.0105g of vitamin B6 reference substance was added to a 10mL volumetric flask, and mobile phase was added to the mark to produce a solution with a concentration of 1.06mg/mL (Stock Solution). The process was repeated with 2mL and 1mL of the Stock Solution to produce solutions with concentrations of 212.00µg/mL (Standard Solution (1)) and 106µg/mL (2). The process was repeated with 1ml of (1) to produce a solution with a concentration of 21.20µg/mL (3). The process was repeated with 1mL and 0.5mL of (4) to produce solutions with concentrations of 10.60µg/mL (5) and 5.30µg/mL (6)
30 Vitamin B6 tablets (1.9104g) were weighed and ground down. An amount approximately equal to 0.1g of vitamin B6 (0.6364g) was added to a 100mL volumetric flask. An appropriate amount of mobile phase was added and sonicated, after which it was diluted to the mark with mobile phase. The mix was then filtered, after which 5mL of the filtrate was added to a 50mL measuring flask, and diluted to the mark with mobile phase (Sample 1). The process was repeated, though with 0.0501g of the vitamin B6 reference substance also added to the powder before the first amount of mobile phase was added (Sample 2)
Standard Solution (3) was injected. It was found to have a retention time of 9.863 seconds and a tailing factor of 0.968 with a column efficiency of 15468. The chromatogram of the Vitamin B6 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 B6 reference substance can be seen on the left. A qualitative RSD% (n=6) of 0.240 and a quantitative RSD% (n=6) of 0.952 were calculated from the values below.
|Name||Retention Time||Peak Area|
Linear Range of the Standard Curve
Standard Solutions (5), (4), (3), (2), and (1) were successively injected once each, the chromatogram of which can be seen on the right
The standard curve of the Vitamin B6 reference substance can be seen on the left. The concentration range was found to be 5.3μg/mL - 212.0μg/mL, the regression equation Y = 5.12459 * X - 5.99461, and the correlation coefficient r=0.9996.
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 5.30mg/L, a peak height mAU of 2.104, a noise of mAU 0.0292, the LOD (mg/L) S/N = 3 was calculated to be 0.221 and the LOQ (mg/L) S/N = 10 to be 0.736.
Sample Measurement Results
Sample 1 was injected, the chromatogram of which can be seen on the left. With a peak area of 509.585, a peak height of of 41.973, and a column efficiency of 15405, the ingredient content was calculated to be 10.1mg/tablet, using the content calculation formula below.
Vitamin B6 Content (mg/tablet) =
[(AS + 5.99461] / 5.12459 * 50 / 5 * 100 / 10 / 1000
Where AS is the peak area of Vitamin B6 in the Vitamin B6 tablet solution.
Sample Measurement Repeatability
Sample 1 was injected was injected 6 times. The qualitative and quantitative chromatogram of the Vitamin B6 sample solution can be seen on the right. A qualitative repeatability RSD% (n=6) 0.078 and a quantitative repeatability of RSD% (n=6) of 0.630 were calculated from the values below.
|Name||Retention Time||Peak Area|
Rate of Recovery
Which is not specified in the original Solution (2) 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 + 5.99461) / 5.12459 - (AS + 5.99461) / 5.12459] * 50 / 5 * 100 / 1000 / CA * 100%
AR is the peak area of the Vitamin B6 tablet solution after adding the Vitamin B6 standard solution,
AS is the average peak area of the Vitamin B6 tablet solution across the 6 injections, and
CA is the concentration of the Vitamin B6 standard substance added to the Vitamin B6 tablets (mg) These were the only units given, just milligrams
Of the three injections, the AR were given at 753.544, 755.387, and 754.004. All three shared an AS of 505.456, and a CA of 50.1 (Which is given as µg/ml in the chart), giving the calculated rate of recoveries as 96.6%, 97.3%, and 96.8%, with an average rate of recovery of 96.9%.
The L-3000 HPLC is capable of quickly and efficiently carrying out the Vitamin B6 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.