Enhanced analytical flexibility: simultaneous normal and reversed phase chromatography

Enhanced analytical flexibility: simultaneous normal and reversed phase chromatography — application summary

Importance of the topic

The combined analysis of fat‑soluble and water‑soluble vitamins in a single workflow addresses a common laboratory challenge: different vitamins require orthogonal chromatographic chemistries for robust separation and accurate quantification. A platform capable of running normal phase (NP) and reversed phase (RP) separations in parallel increases throughput, reduces instrument footprint, and preserves method integrity when working to compendial methods such as USP monographs.

Goals and study overview

The application evaluated the Thermo Scientific Vanquish Flex Duo UHPLC System in a hybrid configuration (one flow path run in NP, the other in RP) to:

• Simultaneously analyze vitamin D3 (fat‑soluble) by NP chromatography and vitamins B1, B2, B3, and B6 (water‑soluble) by RP chromatography according to USP monographs.
• Demonstrate system suitability, precision, and quantitative agreement with label claims for a pregnancy multivitamin capsule.
• Showcase practical sample preparation approaches consistent with USP methods.

Methodology and sample preparation

Overview of analytical approach:

• NP path: Vitamin D3 (cholecalciferol) analysis following USP {581} Vitamin D Assay. Standards and system suitability solutions prepared in n‑hexane; sample extraction included dimethyl sulfoxide (DMSO) and n‑hexane with reflux at 60 °C under N2 to generate and separate precholecalciferol for resolution assessment.
• RP path: Water‑soluble vitamins analyzed according to USP Water‑Soluble Vitamins, Method 2. Aqueous phosphate/hexanesulfonate buffer (pH 3.5, with acetonitrile modifier) served as mobile phase; detection at 254 nm for B1, B3, B6 and 280 nm for B2.
• Sample workup: Capsule contents were combined, extracted with appropriate solvent mixtures (NP: DMSO/hexane reflux; RP: aqueous/organic extraction and filtration), filtered (0.45 µm nylon), and diluted to volumetric endpoints prior to injection.

Chromatographic conditions (concise)

Key chromatographic choices included amino and reverse‑phase Hypersil GOLD columns (150 × 4.6 mm, sub‑5 µm packings), isocratic/gradient conditions adapted to the USP methods, flow rates appropriate for 1 mL/min and 100 µL injection volumes for NP versus 10 µL for RP, and room/controlled thermostating for columns and autosampler to ensure stability.

Used instrumentation

The following Vanquish Flex Duo components and accessories were used:

• Vanquish Flex system base with Dual Pump F
• Dual Split Sampler FT1 and Column Compartment H
• Variable Wavelength Detector F and Diode Array Detector HL (Standard Flow cells and LightPipe options)
• Normal Phase (NP) Kit for Flex systems to allow NP solvent compatibility and independent flow paths
• Chromeleon CDS (Thermo Scientific Chromeleon Software 7.3.2) for data acquisition and processing

Main results and discussion

System performance and suitability:

• Vitamin D3 (NP): Standard solution injections (n=6) produced an average peak area with RSD = 0.13% (well under USP limit of 3%). System suitability mixture resolved cholecalciferol from precholecalciferol with Rs = 12.8 (USP requirement Rs ≥ 10).
• Water‑soluble vitamins (RP): Standard solution injections (n=6) yielded peak area RSDs well below the USP requirement of 2% for each analyte (highest observed RSD: pyridoxine 0.16%).
• Quantification in capsules: Six replicate injections of sample solutions produced calculated amounts normalized to label claims as follows — niacinamide 103.8%, pyridoxine 96.0%, riboflavin 112.5%, thiamine 100.0%. All values fall within the USP acceptance window of 90–125%.

Discussion points:

• The dual‑flow‑path architecture (independent pumps, injectors, columns, and detectors) effectively prevents cross‑contamination (separate needle wash solutions) and enables true parallel analysis within a single physical platform.
• Use of the NP kit requires attention to long equilibration times and compatibility of wetted parts with NP solvents; consult vendor guidance for long‑term solvent exposure.

Benefits and practical applications

The combined NP/RP solution offers concrete laboratory advantages:

• Increased sample throughput — parallel separations reduce total analysis time per sample set compared with sequential runs on separate instruments.
• Reduced capital and bench footprint — one platform performs orthogonal chemistries.
• Compliance with compendial methods — analyses followed USP monographs, limiting the requirement for full revalidation when methods are applied as written.
• Robust QA/QC capability — demonstrated low RSDs, adequate resolution, and quantitative agreement with label claims suitable for routine QC of dietary supplements and similar matrices.

Future trends and potential applications

Expected directions and uses for hybrid NP/RP UHPLC workflows:

• Extension to broader and more complex nutritional matrices (fortified foods, complex supplement blends) where multiple analyte classes require orthogonal separations.
• Integration with mass spectrometric detectors for improved selectivity and sensitivity, especially for trace fat‑soluble analytes or isobaric interferences.
• Automation of sample preparation tailored to parallel workflows to further increase throughput and reduce hands‑on time.
• Standardization of NP solvent‑compatible consumables and long‑term materials to expand NP usage in routine labs.

Conclusion

Deploying the Vanquish Flex Duo UHPLC System with an NP kit enabled simultaneous, compendially compliant analysis of vitamin D3 (fat‑soluble) and vitamins B1, B2, B3, B6 (water‑soluble) from pregnancy multivitamin capsules. The platform delivered excellent system suitability metrics, tight precision, and quantitative results within USP acceptance criteria, illustrating how parallel NP and RP separations on a single instrument increase analytical flexibility and laboratory efficiency.

References

1. United States Pharmacopeia (2021). Dietary Supplement Monographs, Water‑Soluble Vitamins Preparation. USP‑NF. Rockville, MD: United States Pharmacopeia.
2. United States Pharmacopeia (2023). General Chapter {581} Vitamin D Assay. USP‑NF. Rockville, MD: United States Pharmacopeia.