Enhancing the LC-MS/MS Analysis of B-group Vitamins with MaxPeak High Performance Surfaces Technology

Waters MaxPeak High Performance Surfaces (HPS) provide an effective solution to mitigate interactions between analytes and metal surfaces in liquid chromatography. This application note investigates the effects of the MaxPeak HPS on the LC-MS/MS analysis of B-group vitamins and demonstrates the key benefits of using the MaxPeak HPS in the simultaneous analysis of B-group vitamins in energy drinks and vitamin B complex dietary supplements. The key benefits observed include high response, improved sensitivity, less peak tailing, better calibration linearity, and no carry-over compared to the stainless-steel surfaces in a conventional LC system setup. Greater sensitivity (3–10 times) was observed for riboflavin, thiamine, nicotinamide, flavin mononucleotide, pyridoxal 5’-phosphate, and 5-methyltetrahydrofolate using the Waters ACQUITY Premier Solution. The analytical performance (accuracy and repeatability) of the simultaneous LC-MS/MS analysis of B-group vitamins in energy drinks and vitamin B complex dietary supplements are also presented. The ACQUITY Premier Solution (system and column) showed clear advantages, such as improved sensitivity, less peak tailing, better calibration linearity, and no carry-over issue, over the conventional liquid chromatography solution for the analysis of B vitamins.

Benefits

• Waters ACQUITY Premier Solution improves LC-MS/MS analysis of B vitamins
• 3 to 10 times better sensitivity were observed for six B vitamins using the MaxPeak HPS than the conventional stainless-steel surfaces
• Higher response, less peak tailing, and less carry-over are observed with Waters ACQUITY Premier Solution

The B-group vitamins include eight B vitamins, which are thiamine (B₁), riboflavin (B₂), niacin (B₃), pantothenic acid (B₅), pyridoxine (B₆), biotin (B₇), folate (B₉), and cyanocobalamin (B₁₂). Each B vitamin has different forms (or vitamers). Recently, new vitamin B vitamers, such as flavin mononucleotide (FMN) and pyridoxal 5’-phosphate (PLP), have been formulated in dietary supplements. These phosphate containing compounds are often called “native” or coenzymated B vitamins and are believed to be readily absorbed by the human body. The structures of the B vitamins and their vitamers are shown in Figure 1. The interactions between phosphate containing compounds and stainless-steel surfaces and their detrimental effects on high performance liquid chromatography (HPLC) have been reported.^1–2 These interactions can cause severe peak tailing, reduced peak height, and/or carry-over, which lead to inaccurate and unreliable results in HPLC analysis. There are a few workarounds available to address this analyte metal interactions issue. However, these workarounds have their own limitations,³ and a better approach is needed.    

Waters MaxPeak High Performance Surfaces (HPS) technology, which are implemented in the Waters ACQUITY Premier Solution (including system and columns), provides an effective way to mitigate the analyte metal interactions. Dramatic improvements have been observed using the MaxPeak HPS in HPLC analyses for organic acids, oligonucleotides, peptide, glycans, and phospholipids.^4–7 This application note investigates the effects of the MaxPeak HPS on the analysis of B-group vitamins using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The advantages of applying the MaxPeak HPS to the analysis of B vitamins, including those non-phosphate containing B vitamins, are highlighted in the simultaneous LC-MS/MS analysis of B-group vitamins in energy drink and dietary supplement samples. 

All sample preparation was conducted in dimmed light environment and with amber glassware.   

Standard Preparation

Thiamine, thiamine pyrophosphate (TPP), riboflavin, flavin mononucleotide (FMN), nicotinic acid, nicotinamide, pantothenic acid, pyridoxine, pyridoxamine, pyridoxal, pyridoxal 5’-phosphate (PLP), biotin, folic acid, 5-methyltetrahydrofolate (5MTHF), cyanocobalamin (CN B₁₂), methylcobalamin (Me B₁₂), adenosylcobalamin (Aden B₁₂) were purchased from Sigma-Aldrich (St. Louis, MO). Stable isotope labeled B vitamins: ¹³C₅, ¹⁵N-folic acid, ¹³C₄, ¹⁵N₂-riboflavin, ¹³C₄ -thiamine, ²H₄ -biotin, ¹³C₆, ¹⁵N-pantothenic acid, ²H₂ -pyridoxine, and ²H₄ -nicotinic acid were purchased from Isosciences (isosciences.com, Ambler, PA).  Individual B vitamin stock solutions were prepared by dissolving individual vitamins with deionized water at concentrations of 0.5 mg/mL (recorded to 3 significant figures) except riboflavin and biotin, which were prepared as a mixed stock solution at 0.05 mg/mL (recorded to 3 significant figures) for each riboflavin and biotin standard. The folic acid stock solution may need a small amount (one drop or two) of ammonium hydroxide solution (28–30% NH₃) to assist the dissolution. The PLP stock solution was heated to 50 °C to assist dissolution. 

Three intermediate B vitamin mix solutions were prepared by diluting aliquots of the individual stock solutions with deionized water as follows, one intermediate standard mix solution of CN B₁₂ (0.1 mg/mL) and Me B₁₂ (0.1 mg/mL), one intermediate standard mix solution of thiamine (0.01 mg/mL), pantothenic acid (0.1 mg/mL), pyridoxine (0.01 mg/mL), nicotinamide (0.1 mg/mL), nicotinic acid (0.1 mg/mL), and pyridoxal (0.01 mg/mL), and one intermediate standard mix solution of folic acid (0.1 mg/mL) and 5MTHF (0.1 mg/mL). The working standard solutions were prepared from these intermediate B vitamin mix solutions and the remaining individual standard stock solutions by mixing and dilution  with deionized water to various concentration levels.   

The individual stable isotope labeled B vitamin standard stock solutions were prepared in a similar way as previously described for the individual B vitamin stock standards. A mixture of all stable isotope standard stock solution was prepared from these individual stock solutions by mixing appropriate volumes of the individual stock solutions with deionized water to obtain a concentration of 0.02 mg/mL for each standard except ¹³C₅, ¹⁵N - folic acid which was prepared at 0.2 mg/mL. This stable isotope standard mixture stock solution was diluted with deionized water 10 times to make an intermediate stable isotope mixture solution, which was later used to fortify the working standard solutions and the sample solutions as internal reference standards at 0.1 μg/mL, except ¹³C₅, ¹⁵N - folic acid which was added at 1 μg/mL, in the final solutions.

Sample Preparation  

The energy drinks (ED) from major brands, such as Red Bull, Monster Energy, and Rockstar, were purchased from a local store. They were filtered through a 0.45 µm GMF membrane syringe filter. An aliquot of the filtrate was analyzed without dilution for low concentration B vitamin (CN B₁₂). Another aliquot of the filtrate was diluted with deionized water 50 times (1:50 by volume) and analyzed for other B vitamins. A vitamin B complex dietary supplement (DS, liquid form) was also tested. 50 µL of DS was taken and diluted with 200 mL deionized water first, then filtered through a 0.45 µm GMF membrane syringe filter and further diluted with deionized water at different ratios (0.95:1, 1:10, 1:100 by volume) prior to the analysis.

The LC-MS/MS conditions used in the AOAC Official Method 2015.14 for simultaneous determination of total vitamins B₁, B₂, B₃, and B₆ in infant formula and related nutritionals⁸ were used in this study with two minor modifications. The pH of the mobile phase A was adjusted to pH 5.0 to ensure consistent results. The MRM transitions and MS detection parameters for 18 vitamins, including the additional vitamins B₅, B₇, B₉, and B₁₂ were optimized for the Xevo TQ-S micro MS System. 

Effects of the MaxPeak HPS on the Analysis of B vitamins

Two LC system setups were used in the comparison study of the effects of the MaxPeak HPS on the analysis of B vitamins. One comprised of an ACQUITY Premier System and an ACQUITY Premier BEH C₁₈ (1.7 μm, 2.1 x 100 mm) Column (referred to as the HPS setup), and the other one comprised of an ACQUITY UPLC H-Class PLUS System and an ACQUITY UPLC BEH C₁₈ (1.7 μm, 2.1 x 100 mm) Column (referred to as the SOP setup). These two LC systems were identical to each other except that the HPS setup had MaxPeak HPS while the SOP setup had conventional stainless-steel surfaces. The same Xevo TQ-S micro MS System was used in these two systems to minimize instrumental variables in the comparison study. 

Increased Response with the MaxPeak HPS

Higher peak intensities and larger peak areas were observed for majority of the 18 vitamins with the HPS setup. Figure 2 shows comparison plots of peak areas from 7 replicate injections of a standard mix solution (at a concentration of 1 μg/mL) with both LC setups. The data were obtained when the systems and columns were fresh, i.e., no B vitamins have ever been injected onto the systems. One can see that the same or greater peak areas were obtained with the HPS setup for all 18 vitamins than those with the SOP setup. Figure 3A shows comparison of the chromatograms of the FMN, Thiamine, PLP, and Pantothenic acid from the first injections of the same standard mix solution on both LC setups. The peak intensities of these compounds were significantly larger with the HPS setup. This large difference in LC-MS/MS response was attenuated after repeated injections of B vitamins (as the surfaces, especially the stainless steel surfaces, were conditioned by exposure to the analytes), but was still evident after extended use of the LC-MS System (see Figure 3B). Figure 3B shows a comparison of B vitamin chromatograms for a DS sample obtained on both LC setups. Over a hundred injections were already made on both LC setups when the chromatograms in Figure 3B were collected. Less difference in peak intensity for these B vitamins were observed, but the difference in peak height were still evident between the HPS and the SOP setups. Also, severe peak tailing was still observed for thiamine with the SOP setup (Figure 3B). 

Improved Sensitivity with the MaxPeak HPS

Higher sensitivity was also found with the HPS setup than the SOP setup in the LC-MS/MS analysis of B vitamins. The limit of quantitation (LOQ) was estimated using the working standard mix solutions at signal to noise (S/N) ratio of 10. Table 1 shows the LOQ values and the calibration results obtained with both LC setups for the simultaneous B vitamin analysis for ED and DS samples. The LOQ with the HPS setup shows the same or better LOQs than those with the SOP setup. Six B vitamins show 3–10 times improvement in LOQ with the HPS setup. Better calibration linearity (R²) was also observed with the HPS setup (Table 1).  

No Carry-over with MaxPeak HPS

Figure 4 shows a comparison of the B vitamins LC-MS/MS chromatograms obtained with the HPS and the SOP system setups in a carry-over study. Residual peaks were found in the blank injection on the SOP system setup for riboflavin, pyridoxal, 5MTHF, and MeB₁₂ while no residual peak was found on the HPS system setup. 

Analysis of Energy Drink and Dietary Supplement Samples

The B vitamins in ED and vitamin B complex DS were analyzed using the HPS setup coupled with the Xevo TQ-S micro MS System. Figure 5 and 6 show the B vitamin chromatograms for ED and DS. Not all 18 B vitamins were formulated in these samples, only those B vitamins present in the samples were analyzed.

Table 2 shows the analysis results and the labeled or declared values of B vitamins in ED samples. The accuracy of the LC-MS/MS analysis was assessed by spiking experiment on ED1 sample. Recovery of 91% to 107% were obtained for these B vitamins. Comparing to the labeled values, the determined B vitamin values are in the range of 90% to 124% of the labeled values for ED1 and ED2, and 138% to 180% for ED3. Normally, 25–50% overage in fortified nutrients in food products is commonly used. The relative high B vitamin contents in ED3 indicates excess overage in this product. The analysis repeatability were below 7% RSD for most B vitamins, except that higher RSD (up to 17%) for cyanocobalamin and one result (11%) for nicotinamide in ED2. The high RSD for cyanocobalamin can be explained by the extremely low concentration of cyanocobalamin (near LOQ) in ED1 and ED3. Table 3 shows the analysis and spiking experiment results for a vitamin B complex DS. Excellent recovery for 12 vitamins were obtained (78%–122%). The repeatability of these B vitamins was within 7% RSD in general.

This application note investigated the effects of the MaxPeak HPS on the LC-MS/MS analysis of 18 B-group vitamins and showed lasting benefits of using MaxPeak HPS over conventional stainless-steel surfaces for the B vitamins analysis. The key benefits that were found in the LC-MS/MS analysis of B vitamins include higher responses, less peak tailing, improved sensitivity, better calibration linearity, and less carry-over. These benefits were significant at the initial instrument use stage. After extensive use of the LC system and column, the difference between MaxPeak HPS and convention stainless-steel surfaces became less significant but was still evident as observed in the analysis of B vitamins in commercial ED and vitamin B complex DS. Method validation with ED and DS samples showed excellent accuracy and repeatability. The Waters ACQUITY Premier Solution exhibited clear advantages over conventional LC solution in the sensitivity, accuracy, and precision for the analysis of B-group vitamins.

1. Wakamatsu, A.; Morimoto, K.; Shimizu, M.; Kudoh, S. A Severe Peak Tailing of Phosphate Compounds Caused by Interaction with Stainless Steel Used for Liquid Chromatography and Electrospray Mass Spectrometry. J. Sep. Sci. 2005, 28, 1823–1830.
2. Asakawa, Y.; Tokida, N.; Ozawa, C.; Ishiba, M.; Tagaya, O.; Asakawa, N. Suppression Effects of Carbonate on the Interaction between Stainless Steel and Phosphate Groups of Phosphate Compounds in High-Performance Liquid Chromatography and Electrospray Ionization Mass Spectrometry. J. Chromatogr. A 2008, 1198–1199, 80–86.
3. Lauber, M.; Walter, T. H.; DeLano, M.; Gilar, M.; Boissel, C.; Smith, K.; Birdsall, R.; Rainville, P.; Belanger, J.; Wyndham, K. Low Adsorption HPLC Columns Based on MaxPeak High Performance Surfaces. Waters White Paper, 720006930EN 2020.
4. Birdsall, R. E.; Kellet J.; Ippoliti, S.; Ranbaduge, N.; Shion, H.; Yu, Y. Q. Increasing Chromatographic Performance of Acidic Peptides in RPLC-MS-based Assays with ACQUITY Premier featuring MaxPeak HPS Technology. Waters Application Notes, 720007003EN 2020.
5. Boissel, C.; Walter, T. H. Improved Peak Shape and Wide Selectivity Range with ACQUITY Premier Columns. Waters Application Notes, 720007014EN 2020.
6. Smith, K. M. and Rainville, P. Utilization of MaxPeak High Performance Surfaces for Improved Separation and Recovery of Analytes Associated with the Tricarboxylic Acid Cycle. Waters Application Notes, 720006727EN 2020.
7. Brennan, K.; Lame, M. L.; Donegan, M.; Rainville, P. D. Improved Oligonucleotide SPE-LC-MS Analysis Using MaxPeak High Performance Technology. Waters Application Notes, 720007019EN 2020.
8. Official Methods of Analysis (2019) 21^st Ed., AOAC INTERNATIONAL, Rockville, MD, Method 2015.14. www.eoma.aoac.org [accessed on June 19, 2020].