Vitamins are necessary organic compounds that must be consumed in modest amounts for the biological system to work properly and support appropriate growth and health. The Greek word “VITAMINE,” which meaning “vital for life,” is where the name vitamin originates. Vitamin’s importance was initially understood in early age, and they are crucial for the body for maintaining its normal growth and lack or loss of any vitamins may cause serious progressing diseases as age increases. To guarantee the quality of food items, it is equally crucial to determine the quantity and rate of vitamin degradation in the food products.

Hence analytical method development for the qualitative and quantitative analysis is required for analysing the concentration of vitamins in final food products during its storage and stability period (Ankali et al., 2015).

Water-soluble and fat-soluble vitamins are the two groups into which they fall. Essential fat-soluble vitamins are A, D, E and K., while ascorbic acid and vitamin B complex, which includes thiamine-B1, riboflavin-B2, niacin- B3 pantothenic acid- B5, pyridoxine-B6, biotin-B7, folic acid-B9 and cyanocobalamin-B12, are water-soluble groups (Li and Chen, 2001).

To find dietary references and daily values for the vitamins is very crucial and tedious for the health professional in early days. Now a days it is calculated by dose response curve and endpoint is detected by the ED₅₀ (Effective Dose) or LD₅₀ (Lethal Dose) and several toxicity studies. The FDA (Food Drug Administration) by using guidelines set daily values for the nutritional labelling as % DV for avoiding the toxic effect and differentiates in gender categories based on age.

Comprehensive pre-treatment techniques, such as physical, chemical, chromatographic, microbiological and biological techniques, are used in the extraction of vitamins. The material being analysed and its concentration determines which approach is best for vitamin analysis. Chromatographic techniques, however, are frequently insensitive to low vitamin concentration levels. For these low-level assays, microbiological techniques are used; however, they are slower, more costly and typically less precise. These days, there is less demand for delicate biological testing.

To put forward a simple, reliable and economical technique for simultaneous recovery of water-soluble category vitamins from food supplement is of great importance in food industry and food manufacturer because vitamins are most unstable and easily deteriorate by the presence of light, heat, presence of oxygen. So, developing a validated method which can identify the exact amount of degradation of each vitamin during its storage. According to the findings of this study, it can be added in greater quantities to the finished product in order to maintain food product quality in accordance with FDA guidelines (Kucukkolbastiet al., 2013).

Vitamin analysis is a tedious process due to its complex chemical structures and its complexity with the matrices. Each vitamin has its own unique properties and due to their unstable nature and most of its activity loses during processing and storage as a result of exposure to the high temperature, oxygen and light. Therefore, a method using HPLC or mass spectrometry can be developed for the rapid quantification of all water-soluble vitamins (Trang, 2013).

Different matrices have been used in literature research to provide appropriate methodologies for the assessment of individual or combined water-soluble vitamins. Among these reported methods most commonly used techniques spectroscopic, chromatographic, chemical or biological and hyphenated techniques like HPLC, GC, MS etc., even though hyphenated techniques like GC-MS is very expensive and sensitive, its use in developing stage is risky and required expert personalities for handling, so the HPLC is used worldwide as it involves less sample pre-treatment and easy to maintain, environment friendly and less complicated for the analysis of wide variety compounds.

Since these water-soluble vitamins can be analysed by many other types of method and by using different matrices depending upon the formulation and amount present in that formulation and even from blood plasma, urine etc. each method and each matrix have different condition for getting better resolution and pre-treatment process (Zafra:Gómezet al., 2006).

There are many recent advances in analytical methods of Water-soluble vitamins uses HPLC/DAD, HPLC-MS/MS and RP-HPLC/ELSD. However, there are certain limitations still exist in the methods like few are not able to quantify all 8 vitamin B-complex range in single injection run and require multiple runs at different wavelength for quantification of all vitamins (Abdelfatahet al., 2024; Wazedet al., 2022; Saptariniet al., 2022; Fatimaet al., 2019; Rakusa et al., 2021; Faraz et al., 2019; Klejduset al., 2004; Papadoyanniset al., 1997; Matteva et al., 2023) few methods use derivatization or solid phase extraction techniques which make them time consuming and even more complex, few techniques employ buffers as mobile phase components, resulting in longer processing times and shorter column lives (Ziaet al., 2023; Hosainet al., 2021). HPLC-MS/MS are highly sensitive and reliable techniques but cost is the main factor while considering the routine analysis.

To address all these challenges this research aims to develop a method, which is simple, sensitive, robust and capable for simultaneous estimation of water-soluble vitamins (B1, B2, B3, B5, B6, B7, B9 and B12) in a single method at single wavelength (210 nm) utilising a PDA detector in formulated food product and validate as per ICH guideline. Three different brands of Food Products (HFD) samples were purchased from the local market and tested with the validated method to compare with label claim.

Analytical determination was held using Shimadzu HPLC system (LC-2030C3D) with Phenomenex C18 column (250×4.6 mm), 5 µ, Flow rate 0.8 mL/min, Wavelength used for detection 210 nm, Column temperature 30ºC, pump consisting of five line degassing unit with gradient elution of four solvents with a maximum pressure of 2244 MPa, autosampler of needle injection mode of capacity 0.1-100 µL, oven temperature ranges from 4ºC to 60ºC and detector used is PDA detector of wavelength range from 190-700 nm, flow cell capacity of 12 µL, with low noise level base line detection.

The water-soluble standards for reference standard were procured from Sigma Aldrich Company and Supplier Company. The purity of thiamine mononitrate (B1) is 99%, riboflavin (B2) is 97.9%, niacinamide (B3) is 99.21%, calcium pantothenate (B5) is 100.0158%, pyridoxine (B6) is 99.70%, biotin (B7) is 99% folic acid (B9) is 97.10%, cyanocobalamin (B12) is 99.20%. These reference standards are in powder form and stored in dark cool condition to prevent degradation from light and sunlight. Other than these vitamins, materials used include orthophosphoric acid (thermo Fischer), acetonitrile (Qualigens), milli-Q water.

Malt extract (liquid form), Sugar powder form, Puffed rice powder, Soya flour, Casein, coco butter, Vitamin premix, Mineral premix (salt form).

To prepare the diskettes formulation with the above-mentioned ingredients, oil is heated and to that small quantity of water is added. To this oil water mixture all the powder ingredients like soya flour, casein, vitamin premix, mineral premix. Then add crispy items like puffed rice powder and sugar powder. Mixed well and passed it through 8 mesh size for producing granules. These granules are dried in oven by keeping at 60ºC for 2 or 3 hr. Again, pass it through small mesh size to produce fine powder. Finally added flavours and transferred this powder mass into punching machine. The final diskettes are dried in oven at 60ºC.

The mobile phase contains mixture of two water miscible solvents- 0.1% orthophosphoric acid (Fischer scientific HPLC grade) in water (A) and 80% acetonitrile (Qualigens HPLC grade) with 0.1% orthophosphoric acid (B). The gradient programme was optimized-A/B-98: 02 for 5 min, ramped to A/B-72: 28 in 17 min and held constant at A/B-72: 28 for 3 min. Total run time were 35 min for all 8 vitamins.

Each standard vitamin (B1, B2, B3, B5, B6, B7, B9 and B12) was prepared by putting around 5 mg in 100 mL flask separately and 50 mL of HPLC grade water was added then the standard was sonicated for 5 min and volume was made up-to 100 mL by using same diluent. To identify individual peaks of vitamins these individual standards were injected in HPLC.

After the retention time identification of each of individual vitamins, mixed standard solution of all vitamins was used further.

The mixed stock solution was made with 100 mg of each standard vitamin in 100 mL flask and HPLC grade water to get 1 mg/mL concentration and different dilution were made by using the stock solution. To prepare glass vials for HPLC analysis, supernatant was directly poured into them.

The diskette sample was made powder by using pestle and mortar, the powdered sample was weighed approximately 1 g in a 100 mL volumetric flask, to which 50 mL of diluent (HPLC grade water) was added. Sample was then sonicated for 5 min, the volume was adjusted up-to 100 mL and filtered through a 0.45 µm PVDF syringe filter in glass vial and injected into chromatographic system (HPLC).

Validation of Method was done using the ICH recommendations’ metrics, which included robustness, system suitability test, Limit of Quantification (LOQ), Limit of Detection (LOD), specificity, range, linearity, precision and accuracy.

An analytical procedure is deemed linear by the ICH criteria if it is able to yield test results that are precisely proportionate to the analyte concentration in the sample. By injecting the sample in duplicate and by making a calibration curve by graphing concentration vs. area, the linearity of water-soluble vitamins was ascertained. By ensuring that the correlation coefficient r² was more than 0.99, linearity of the method was verified.

According to the ICH precision guideline, the analytical procedure expresses how closely several measurements taken from several sample under the given conditions agree with one another. There are different types of precision: intraday and interday and repeatability. By injecting samples 6 times and comparing the results to the standard, the precision can be ascertained. The 6 injections’ RSD was computed.

The laboratory’s intra-day precision was represented within a day variation, demonstrating results that are in close proximity to a set of measurements derived from several samplings. Precision across same condition is expressed by repeatability. Repeatability is achieved by injecting the 6 runs of standard and samples. Intermediate precision can be demonstrated by conducting an experiment in a separate lab by a different individual at a different time. Through the use of different instruments for the same experiment, the intermediate precision demonstrates the method’s robustness. The acceptance criteria for all, was maximum 3.0%. RSD.

Agreement degree between the value that is recognised as either an acceptable reference value or a conventional actual value is expressed by the accuracy of an analytical method. After spiking the sample with 50%, 100% and 150% of a specified amount of standard, the percentage recovery of the vitamins was used to calculate the method’s accuracy. In order to determine the ultimate RSD and percentage recovery, three runs of unspiked samples, 50% spiked samples, 100% spiked samples and 150% spiked samples were examined. An accuracy acceptance criterion was 80-120% with maximum 3.0% RSD.

A specific analytical method’s detection limit is the lowest concentration of analyte in a sample that can be detected but not accurately measured. There are numerous methods for figuring out the detection limit, including visual inspection, the noise to signal ratio and standard error graphing with a standard calibration curve.

σ: Standard deviation derived from response,

S: Slope derived from calibration curve,

(ICH Q2 R1 guidelines).

In analytical method, the limit of quantification is the lowest practical concentration of an analyte in a sample that can be quantitatively measured with adequate precision and accuracy. A parameter of quantitative testing called the limit of quantification is particularly helpful for identifying contaminants and/or degradation products at low chemical concentrations in sample matrices.

σ: Standard deviation derived from response,

S: Slope derived from calibration curve,

(ICH Q2 R1 guidelines).

The range of the method is the range of analyte concentrations in the sample between which the analytical procedure has been shown to have an appropriate degree of linearity, accuracy and precision. The sample concentration was determined by taking its 25% to 125% (2.5-15 mg/mL). The standard concentration was 0.0005 mg/mL to 0.6 mg/mL. The range was determined by comparing the maximum high concentration and lowest concentration of the standard to the concentration that will be present in the finished product.

Indicating its dependability under typical operating conditions, robustness is a measure of its ability to withstand little but intentional variations in method parameters. We evaluated the method’s robustness in this instance by varying the vitamin’s wavelength and flow rate. Though the retention period has altered, the approach still yields the same results.

By minimally altering the main procedure portion without altering the solvent or analyte, ruggedness checking can be carried out.

In the developing stage, system suitability tests are carried out to demonstrate that the system can carry out the suggested technique for the identification of specified standards and samples. Theoretical plates, Capacity factors, Tailing Factor and Resolution are among the key parameters for system suitability.

The purpose of the specificity test is to quantify the analyte of interest in the presence of other components that may be anticipated to exist. These typically contain matrixes, degradants and contaminants. The specificity of a method is assessed using a placebo and solvent.

The correlation coefficient was identified by plotting the area v/s concentration. The linear regression and correlation coefficient were found to be >0.99 for all the water-soluble vitamins.

The most common technique for determining the LOD and LOQ is the Signal to Noise ratio (S/N), which is 3:1 for the LOD and 10:1 for the LOQ (Table 1). Other methods include visual evaluation, standard deviation of response and slope. Software can also be used to calculate LOD and LOQ and report results.

By adding a known quantity of standard to the sample, one can obtain reliable results by measuring the percentage of recovered data using the accuracy parameter. The range used for detection was 80-120% and the accuracy within the concentration range met the acceptance criteria with a percentage RSD inside the limit.

Intraday was performed by injecting 6 samples replicates on the same day whereas interday was done on a different day. All results were found within max. 3.0% RSD.

System suitability ensures that system is suitable to perform the quality testing without any interference. The result found passes the system suitability criteria (Table 2).

The validation parameter is crucial, because despite our deliberate changes to the parameters, the outcome ought to be the same. The wavelength and flow rate per minute are the two most often changed parameters in the process. Wavelength change was performed at 208 and 212 nm whereas change in flow rate was performed at 0.7 mL/min and 0.9 mL/min. The RSD of all robustness parameters was found to be under the 3.0% limit.

Peak of individual vitamin in the sample was confirmed by comparing with Retention Time (RT) by overlying with standard vitamin and by comparing with standard UV spectra. The peak purity angles of all the peaks were found to be lower than their respective peak threshold, confirming the purity of peaks.

Vitamins quantification in Formulated food sample and in Market sample are shown in Tables 5 and 6 respectively.

This analytical technique is validated according to ICH guidelines Q2(R1) for all the criteria. The developed method can elute all 08 vitamins B1, B3, B6, B5, B9, B12 B2 and B7 as shown in Figure 1. However, in the sample due to absence of vitamin B7, B9 and B12, the rest of the vitamins validation was carried out. The formulated food sample as well as market samples were tested as per the procedure mentioned earlier at the concentration of 10 mg/mL as shown in Figure 2 for which values were found within 90-110% of label claim. This demonstrated the applicability of proposed method for analysing the water-soluble B-Complex vitamins in different finished food products.

Figure 1:
HPLC Chromatogram of standard water soluble vitamins 1) B1 (RT 2.813 min) 2) B3 (RT 4.001 min) 3) B6 (RT 5.951 min) 4) B5 (RT 17.212 min) 5) B9 (RT 20.270 min) 6) B12 (RT 22.241 min) 7) B2 (RT 24.300 min) 8) B7 (RT 25.434 min).

Figure 2:
HPLC Chromatogram of vitamins (water soluble) in Formulated Food Supplement. B1 (RT 2.793 min) 2) B3 (RT 3.985 min) 3) B6 (RT 5.967 min) 4) B5 (RT 17.269 min) 5) B2 (RT 24.314 min).

This research addressed the limitation of the available methods as we were able to analyse all vitamin B-complex in a single method at single wavelength. We have simplified the process of sample preparation by using only purified water as a diluent compared to complex sample preparation like solid phase extraction or derivatization. The current method has been developed and validated using the simple mobile phase that does not contain any buffer system, which makes the process time saving and extending column life compared to existing method. With recovery rates of 80-120%, the method ensures high accuracy in the food matrix. This research removes barriers to wider applications by demonstrating that the approach is applicable to a variety of food matrices and is not just restricted to designed food products. Stress degradation study can be explored further to improve the scope of the method in terms of stability indicating under various conditions.

The RP-HPLC method was successfully developed and validated for the determination of all B complex water-soluble vitamins. The method developed was validated by ICH guidelines in terms of accuracy, precision, linearity and specificity. This method shows good resolution between all vitamins in single run and single wavelength which makes it economical as multiple runs can be minimized. The food and pharmaceutical industries could benefit from this method’s increased utility in the future by investigating additional optimization for other complicated matrices or extending its application to liquid and pharmaceutical samples.

The author Tejas Vyas is grateful to Dr. S M Sivaramakrishnan, Head R&D, Zydus Wellness Ahmedabad-380058, Gujarat, India and Faculty of Pharmacy, Nootan Pharmacy College, Sankalchand Patel University, Visnagar for extending the required support and resources to carry out this research work. The authors have no conflict of interest.