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Simultaneous Specific Determination of Caffeine, Riboflavin (Vitamin B2) and Pyridoxine (Vitamin B6) In Non-Alcoholic Beverages, Beverage Concentrates and Syrups Using Isocratic HPLC-UV 

Document #031020-10. Revision 01, 10 March 2020



Caffeine, Riboflavin (vitamin B2), Pyridoxine (vitamin B6).

Scope of The Method

The method is intended for the highly selective determination of caffeine, riboflavin (vitamin B2) and pyridoxine (vitamin B6) in various beverage, beverage concentrate and syrup samples using simple isocratic 400 bar HPLC system with a conventional UV detector.
      The method is capable to determine caffeine in complex matrices that contain water-soluble vitamins (C, B1, B3, B9), preservatives (benzoic and sorbic acids), natural and synthetic dyes, UV-absorbing flavoring agents (vanillin, ethylvanillin, benzaldehyde, etc.), natural polyphenolic compounds including hydroxybenzoic and hydroxycinnamic acids and aldehydes.


Minimum System Requirements
Solvent Delivery System: Isocratic HPLC pump with 400 bar upper back-pressure limit 
Detector: Single programmable wavelength UV detector, or dual wavelength UV detector
Column Oven: Preferable to ensure retention time stability

Compartible with all HPLC systems: Agilent, Shimadzu, Waters, Thermo, etc.

General Information On The Method
Elution Mode: isocratic, Detection: UV, Actual Analysis Time: 5.5 minutes, Back-pressure: typically < 300 bar, HPLC column manufacturer: HELIX Chromatography, USA >> 


Caffeine, Riboflavin (vitamin B2), Pyridoxine (vitamin B6), HPLC, Agilent, Shimadzu, Waters, Thermo Scientific

Fig. 1. Isocratic determination of caffeine, riboflavin, and pyridoxine in the beverage concentrate #1. Detection: 0-3 min UV 270 nm, 3-4 min UV 360 nm, 4-5.5 min UV 300 nm.
1. Caffeine,  2. Riboflavin, 3. Pyridoxine.

Caffeine, Riboflavin (vitamin B2), Pyridoxine (vitamin B6)


The method may require soft adjustment to provide an excellent result for a particular sample. Retention and separation selectivity tuning recommendations are given in the application.

        We can fine-tune method performance to meet your requirements upon request.

        We can develop the complete HPLC method based on this application upon request.

Table of contents

Description of The method  5
Name of The Method 5
Scope of The Method  5
Main Characteritics of The Method 5
Minimum System Requirements 5
Analyte(s) 6
Standard HPLC Conditions 6
Typical Chromatogram(s) 7
Suitable HPLC Column(s) 7
Quickstart Steps 8
Mobile Phase Preparation 8
Column Washing And Storage 8
Column Conditioning 8
Testing The Column Performance 8
Fine-Tuning Retention And Selectivity 9


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