Application ote Food Testing and Agriculture Analysis of Vitamins Using an SF/UPL ybrid System with a Triple Quadrupole L/MS for Quantification ounts 4.6.4...8.6.4....4.6.8...4.6.8 Acquisition time (min 4 ounts 4..4.6.8.. 4. 4.6..4 4 Acquisition time (min Author Edgar aegele Agilent Technologies, Inc. Abstract This Application ote describes the quantitative determination of fat- and water soluble vitamins using an Agilent 6 Infinity II SF/UPL ybrid System with an Agilent 647A Triple Quadrupole L/MS System. A selection of fat-soluble vitamins are separated in SF mode, and a selection of water-soluble vitamins are separated in UPL mode. All vitamins are detected and quantified using the L/MS system. This study demonstrates that the 6 Infinity II SF/UPL ybrid System can determine both fat- and water-soluble vitamins with comparable performance as standalone SF and UPL systems.
Introduction Performing both an SF-based separation and a UPL-based separation of a given complex sample delivers complementary information about the sample content. These separations are truly orthogonal due to their different separation mechanisms, which are based on the interaction of the analytes with completely different fluid media and stationary phases. onversely, it is also possible to analyze samples for analytes, which either perform better under SF separation conditions or under UPL separation conditions to obtain complementary information. For example, a group of fat soluble vitamins can easily be separated under SF conditions, whereas a group of water soluble B-vitamins can be separated under reversed-phase conditions. This Application ote describes the analysis of fat-soluble and water soluble vitamins on the 6 Infinity II SF/UPL ybrid System in SF mode and UPL mode with triple quadrupole mass spectrometry for their quantification. Experimental Instrumentation Agilent 6 Infinity II SF/UPL ybrid System: Agilent 6 Infinity II SF ontrol Module (G4A Agilent 6 Infinity II SF Binary Pump (G478A Agilent 6 Infinity II SF Multisampler (G4767A Agilent 6 Infinity II Diode Array Detector (G7A with high pressure SF flow cell Agilent 6 Infinity II Multicolumn Thermostat (MT (G76A with Agilent InfinityLab Quick hange 4-position/-port four-column selection valve (G47A Agilent 6 Infinity II Quaternary Pump (G7B Agilent 9 Infinity Valve Drive (G7A with -position/-port valve (G4 Agilent 6 Infinity II Isocratic Pump (G7B and SF/MS splitter kit (G49-687 Agilent 647A Triple Quadrupole L/MS with Agilent Jet Stream technology Instrumental setup For the conversion of an SF system to an SF/UPL hybrid system, a quaternary or binary UPL pump is added and connected by a -positon/ port valve, which allows direct switching between SF mode and UPL mode (Figure. The MT is equipped with a 4-position/ port four column selection valve for switching, for example, from a typical SF column to a typical analytical UPL column (not shown in Figure. The central point, outlined in Figure, is the plumbing at the -positon/-port valve. The position of the valve shown in Figure A connects the SF pump and the SF control module to the shared modules of the instrument. After flowing through the autosampler, the column oven with the SF column, and the detector, the stream is connected back to the SF control module for backpressure regulation. In this position, the quaternary UPL pump is connected to waste. After switching the -positon/ port valve to the UPL position, the quaternary pump is connected to the shared modules (Figure B. To maintain backpressure, the SF pump is directly connected to the SF control module. An isocratic pump serves as a make up pump to connect the SF flowpath with the L/MS by a flow splitter. In SF mode, the stream is connected to the first splitter for the introduction of the make-up flow after passing the shared modules. The second splitter divides the flow between backpressure regulation at the SF control module and the MS (Figure A. In UPL mode, the quaternary pump is connected to the shared modules, and the full flow is diverted to the L/MS (FigureB.
A B Isocratic Pump Isocratic Pump Figure. Instrumental setup of the SF/UPL ybrid system with connection to MS. A SF mode with connection to the make-up pump and flow splitting to the MS. B UPL mode with direct flow connection to the MS. olumns SF mode: Agilent ZRBAX SB-8,.,.8 µm (p/n 8897- UPL mode: Agilent ZRBAX SB Aq,. mm,.8 µm (p/n 8897 4 Software Agilent penlab DS hemstation Edition for L and L/MS Systems, revision..7 SR Agilent Massunter Data Acquisition software for triple quadrupole mass spectrometer, version 7.. Agilent Massunter Qualitative software, version 7. Agilent Massunter Quantitative software, version 7. Agilent Massunter MRM and Source optimizer software, version 7. SF method for separation of fat-soluble vitamins Parameter Solvent A Modifier B SF flow Gradient Backpressure regulator (BPR temperature BPR pressure Methanol. ml/min minutes %B. minutes %B 4 minutes %B 6 bar olumn temperature 4 Injection volume. µl Feed solvent ver feed volume µl Feed speed eedle wash MS Make up Methanol, µl/min seconds in methanol Value. ml/min, methanol + % water +. % formic acid UPL method for separation of water-soluble vitamins Solvent A Solvent B Flow rate Gradient Stop time Post time Parameter Value Water + mm ammonium formate +. % formic acid Methanol +. % formic acid.7 ml/min minutes %B 7 minutes 7 %B 7 minutes minutes olumn temperature 4 Injection volume. µl eedle wash seconds in methanol
MS method for SF Source parameters for SF mode: See Table. MRM conditions: See Table for the creation of the dynamic MRM method for the determination of seven fat-soluble vitamins in SF mode. MS methods for UPL mode Source parameters for UPL mode: See Table. MRM conditions: See Table for the creation of the dynamic MRM method for the determination of eight water-soluble vitamins in UPL mode. Table. MS source conditions for SF and UPL modes. MS Source conditions SF Method UPL Method Gas temperature Gas flow 9 L/min 9 L/min Sheath gas temperature 4 Sheath gas flow L/min L/min ebulizer psi psi apillary voltage 4, V, V ozzle voltage, V V EMV + V + V Polarity Positive Positive Table. Precursor ions, product ions, fragmentor voltage, and collision energy used for the setup of the dynamic MRM triple quadrupole method for the determination of the seven fat-soluble vitamins separated in SF mode using the 6 Infinity II SF/UPL ybrid System. ompound RT (min Precursor ion (m/z Fragmentor (V Product ion (m/z ollision energy (V ell acceleration (V Product ion (m/z ollision energy (V ell acceleration (V Menaquinone (K.48 44 4 87 4 8 Phylloquinone (K. 4.7 87 4 7 6 4 α-tocopherol.6 4 6 8 4 7 46 Retinol (A.7 69 9 8 8 Retinyl palmitate.79 69 9 8 Ergocalciferol (D.6 97 7 4 69 4 holecalciferol (D.69 8 8 4 7 8 Table. Precursor ions, product ions, fragmentor voltage, and collision energy used for the setup of the dynamic MRM triple quadrupole method for the determination of the eight water-soluble vitamins separated in UPL mode using the 6 Infinity II SF/UPL ybrid System. ompound RT (min Precursor ion (m/z Fragmentor (V Product ion (m/z ollision energy (V ell acceleration (V Product ion (m/z ollision Energy (V ell acceleration (V Pyridoxamine (B 6.87 69. 87 8 4 Thiamine (B.84 6. 9 44 8 icotinic acid (B. 4 7 8.. icotinamide (B.84 8.. Pantothenic acid (B.. 8 8 9. 8 Biotin (B 7 4.4 4 8 7. 8 Folic acid (B 4.9 44 9. 76 4 yanocobalamin (B.9 678.6 7 9 47 4 4
Samples Seven fat-soluble vitamins: Menaquinone (K, phylloquinone (K, α-tocopherol (E, retinol (A, retinyl palmitate, ergocalciferol (D, and cholecalciferol (D (the final concentrations in the mixture were ppm each in Me, which has been used for the creation of calibration curves by further dilution, as described below. Eight-water soluble vitamins: Thiamine (B, pyridoxamine (B 6, nicotinic acid (B, nicotinamide (B, biotin (B 7, pantothenic acid (B, folic acid (B, and cyanocobalamin (B (the final concentrations in the mixture was ppm each in Me, which has been used for the creation of calibration curves by further dilution, as described below, hemicals All solvents were purchased from Merck, Germany. Fresh ultrapure water was obtained from a Milli-Q Integral system equipped with L-Pak Polisher and a.-µm membrane point of use cartridge (Millipak. hemicals were purchased from Sigma-Aldrich (Germany. Results and Discussion Analysis of fat-soluble vitamins in SF mode As an example of the analysis of fat soluble vitamins in SF mode, a mixture of seven fat-soluble vitamins was used (Figure. This mixture was separated in a short four-minute gradient using a 8 column with methanol as a modifier for. All seven compounds eluted in a time window between. and.8 minutes, and were detected by a triple quadrupole L/MS in dynamic MRM mode (Figure. Vitamins K and K eluted early, but separated between. and. minutes. Vitamins D and D eluted at the end of the gradient, between. and.7 minutes. Menaquinone (K α-tocopherol (E Retinyl palmitate Ergocalciferol (D Phylloquinone (K Retinol (A holecalciferol (D Figure. Structures of the seven fat-soluble vitamins, which were separated under SF conditions using the 6 Infinity II SF/UPL ybrid system. ounts 4.6.4...8.6.4.,4...4.6.8...4.6.8 Acquisition time (min Figure. Separation of seven fat-soluble vitamins ( ppb each in a short gradient under SF conditions with detection by triple quadrupole MS in dynamic MRM mode (see Table. 6 7
For all compounds, calibration curves were created between, and ppb (,,,,,,, and ppb. The limit-of-quantification (LQ was calculated for a signal-to-noise (S/ ratio of, and the limit-of-detection (LD for an S/ ratio of. For instance, the LQ for the early eluting vitamin K was ppb, and for the late eluting vitamin D the LQ was ppb, both with excellent linearities (Figure 4. The typically observed LQs were at or below ppb, the typical retention time precisions were at or below. %, and the typical area precisions below.7 % (Table 4. 4 A R =.9996.8 4.6 A 44. & 8. 44. & 87. Responses ounts.4...8.6 Menaquinone (K.4 4 6 7 8 9, oncentration (ng/ml... Acquisition time (min 4 B 6 R =.999.. B 97. & 69. 97. & 7..8 Responses 4 ounts.6.4...8.6 Ergocalciferol (D.4 4 6 7 8 9, oncentration (ng/ml.4..6.7.8 Acquisition time (min Figure 4. alibration curves of: A Menaquinone (K from, to ppb; A Quantifier and qualifier ions at the LQ of ppb (S/ = ; B Ergocalciferol (D from, to ppb; B Quantifier and qualifier ions at the LQ of ppb (S/ =. Table 4. Results obtained from the seven fat-soluble vitamins separated under SF conditions for retention time and area precision, LD, LQ, and linearity (n =. o. ompound RT (min RT RSD (% Area RSD LD (ppb LQ (ppb Linearity Menaquinone (K.48..79.9996 Phylloquinone (K...8.6.999 α-tocopherol.6..7.7..999 4 Retinol (A.7.7..999 Retinyl palmitate.79.9..999 6 Ergocalciferol (D.6..84.999 7 holecalciferol (D.69..7.9997 6
Analysis of water-soluble vitamins in UPL mode In UPL mode, a mixture of eight water-soluble vitamins contained in the B-complex were analyzed (Figure. The eight compounds inherent in the mixture were separated in a short seven-minute gradient by a 8 SB-Aq reversed phase column with acetonitrile/water as mobile phase (Figure 6. The elution started at.8 and.8 minutes with pyridoxamine and thiamine, respectively. All eight compounds eluted in a time window between.8 and. minutes, with cyanocobalamin as the last eluting compound. + l S Thiamine (B icotinic acid (B icotinamide (B Pantothenic acid (B Pyridoxamine (B 6 S Biotin (B 7 Folic acid (B R o + yanocobalamin (B Figure. Structures of the eight water-soluble vitamins, which were separated under UPL conditions using the 6 Infinity II SF/UPL ybrid System. ounts.. 4.... 4..9.8 4..7.6...4. 4...... ounts 6 8 7 4. 4.4 4.6 4.8...4 Acquisition time (min.. 6 7 8 4 Acquisition time (min Figure 6. Separation of eight water-soluble vitamins ( ppb each in a short gradient under UPL conditions with detection by triple quadrupole MS in dynamic MRM mode (see Table. The inset shows the last three lower abundant ions. 7
alibration curves between, and ppb were created for all eight compounds. The LQ was calculated for an S/ ratio of, and the LD for an S/ ratio of. For instance, the calculated LQ for nicotinic acid was.6 ppb, and for the late eluting vitamin B the LQ was ppb, both with excellent linearities (Figure 7. Responses A A. 4. & 8. R =.999 4. &.. 4 icotinic acid (B ounts..9.8.7.6..4... 4. 4 6 7 8 9, oncentration (ng/ml B R =.9996 8..4..6.7.8 Acquisition time (min B 678.6 & 47. 678.6 & 9. Responses... R o + ounts 7 6 yanocobalamin (B 4 6 7 8 9, oncentration (ng/ml 4.8 4.9.....4 Acquisition time (min Figure 7. alibration curves of: A icotinic acid (B from, to ppb; A Quantifier and qualifier ions at ppb (S/ = ; B yanocobalamin (B from, to ppb; B Quantifier and qualifier ions ppb (S/ =. 8
Some of the observed LQs were at or below ppb, the typical retention time precisions were at or below. %, and the typical area precisions below. % (Table. onclusions This Application ote demonstrates the advantage of the 6 Infinity II SF/UPL ybrid System for the analysis of compounds that are preferably analyzed under SF or UPL conditions, on a single system. Analytical results were delivered in both SF and UPL modes, which are comparable with results from standalone instruments regarding LDs, LQs, and precision and linearity of peak areas and retention times. The values determined using the 6 Infinity II SF/UPL ybrid System with the Agilent 647 Triple Quadrupole L/MS for peak area RSD, retention time RSD, linearity, and LDs and LQs demonstrate the excellent performance of the combined system. Table. Results obtained from the eight water-soluble vitamins separated under UPL conditions for retention time and area precision, LD, LQ, and linearity (n =. o. ompound RT (min RT RSD (% Area RSD LD (ppb LQ (ppb Linearity Pyridoxamine (B 6.87..76..6.999 Thiamine(B.84.4.44...999 icotinic acid (B..6.6..6.999 4 icotinamide (B.84.4.9..9994 Pantothenic acid (B..6..999 6 Biotin (B 7 4.4.9.69.999 7 Folic acid (B 4.9..9.9997 8 yanocobalamin (B.9.8..9996 9
References. aegele, E. rthogonal hromatographic Separations using the Agilent 6 Infinity II SF/UPL ybrid System, Agilent Technologies Application ote, publication number 99 876E, 7.. Maria Rambla-Alegre, M.; Dunkle, M..; Vanhoenacker, G.; David, F.; Sandra, P.; Vollmer, M. Analysis of Antioxidants in vegetable oils using the Agilent 6 Infinity SF/UPL ybrid System with MS detection, Agilent Technologies Application ote, publication number 99 46E, 7. www.agilent.com/chem This information is subject to change without notice. Agilent Technologies, Inc. 8 Printed in the USA, May, 8 99-99E