HILIC ILIC BEH BEH 009 9 0 5 009 ihon Waters K.K. HILIC HILIC HILIC MS Sub-µm 009 ihon Waters K.K.
HILIC? HILIC - Hydrophilic Interaction Chromatography 990 * HILIC (> 80%) *Alpert A. J. J.Chromatogr. 99 (990) 77-96. 009 ihon Waters K.K. HILIC : (> 80%) ESI-MS PPT / PPT LLE SPE 009 ihon Waters K.K.
Waters HILIC HILIC (ph-5) Atlantis HILIC Silica HILIC (ph-8) XBridge HILIC ACQUITY UPLC BEH HILIC 009 ihon Waters K.K. 5 XBridge TM HILIC: : XBridge TM HILIC 009 ihon Waters K.K. 6
ew ACQUITY UPLC BEH Amide : :.7 µm (.5 µm 09 ) : 7.5 µmol/m : % : one ph : - 009 ihon Waters K.K. 7 HILIC 009 ihon Waters K.K. 8
: HILIC 5.5 Retention Map H Retention Factor.5.5.5.5 0.5 H + Cytosine HILIC RP 0 0.5 0 0 0 0 50 60 70 80 90 00 % Acetonitrile HILIC 009 ihon Waters K.K. 9 : actor Retention Fa 6 0 8 6 0 Retention Factor Silica Particle A icotinic acid ortriptyline Cytosine Methacrylic acid 70 75 80 85 90 95 % Acetonitrile 6 icotinic acid ortriptyline 0 Cytosine 8 Methacrylic acid 6 0 BEH Particle B 70 75 80 85 90 95 % Acetonitrile BEH 90% BEH : 0 mm with % H C H CH Methacrylic Acid pka.58 H H Cytosine pka =. ortriptyline pka = 0 CH H H 009 ihon Waters K.K. 0 icotinic Acid pka =.,.8 5
Atlantis HILIC Silica Retention Factor = 0 Atlantis dc 8 H H H H Allantoin 0.50.00.00.00.00 5.00 HILIC Retention Factor =. Atlantis HILIC Silica HILIC 0.50.00.00.00.00 5.00 009 ihon Waters K.K. XBridge TM HILIC XBridge TM C 8 HILIC V 0 =0.5. 6-acetyl morphine HILIC XBridge TM HILIC. morphine V 0 =0.6 0.00.00.00.00 5.00 6.00. Morphine -ß-D-Glucuronide 009 ihon Waters K.K. 6
HILIC : BEH Amide vs. BEH HILIC 0.60 U ACQUITY UPLC BEH HILIC. x 00 mm.7 µm. Methacrylic Acid. Cytosine. ortriptyline. icotinic Acid 0 0.60 ACQUITY UPLC BEH Amide. x 00 mm.7 µm ew BEH Amide HILIC 0 0.00.00.00.00 5.00 6.00 7.00 8.00 90/5/5 AC/EtH/0 mm CH CH in H with % CH CH 0.5 ml/min. 5 C.5 µ 60 µg/ml 75/5 AC/MeH UV 0 nm 009 ihon Waters K.K. BEH Amide vs. BEH HILIC BEH Amide BEH HILIC 009 ihon Waters K.K. 7
UPLC BEH Amide ELSD on ACQUITY UPLC using 0.5mL/min at 85 C Isocratic: 77% Acetone with 5% TEA. x 50mm Columns 0.7µL injection Strawberry Smoothie Hot Cross Buns White Bread Tomato Ketchup Lamb Curry Meal Raisin Bran Cereal 5 Sports Drink Food Sugars 0.00.00.00.00 ) Fructose ) Glucose ) Sucrose ) Maltose 5) Lactose (mg/ml each) 009 ihon Waters K.K. 5 : ( methanol isopropanol) HILIC / ph *; ph 5*; 0.% ph.5* 0.% ph.8* 0mM 0.% HILIC MS ph ph Canals I.; umada F. Z.; Roses M.; Bosch E. J. Chromatogr. A. 9 (00) 9-0. 009 ihon Waters K.K. 6 8
HILIC : Water Strongest Methanol Ethanol Isopropanol Primary Solvent Acetonitrile Weakest 009 ihon Waters K.K. 7 : 90:0 AC:H 0.00.00.00.00 5.00 6.00 7.00 8.00 90:5:5 AC:H :MeH 0.00.00.00.00 5.00 6.00 7.00 8.00 90:5:5 AC:H :EtH 90:5:5 AC:H :IPA 0.00.00.00.00 5.00 6.00 7.00 8.00 0.00.00.00.00 5.00 6.00 7.00 8.00 0 mm ammonium acetate with % acetic acid Analytes: : methacrylic acid : cytosine : nortriptyline : nicotinic acid 009 ihon Waters K.K. 8 9
: ph tion to e (k') Relative Retent Acenaphthene.6. 0.8 0. H H salicylic acid BEH Amide BEH HILIC 0 6 8 0 mobile phase ph H H H ion to e (k') Relative Retent Acenaphthene.0 80 8.0.0 H ascorbic acid (Vit C) BEH Amide BEH HILIC 0 6 8 0 mobile phase ph Relative Retention to Acenaphthene (k') 6.0.0.0 nicotinic acid BEH Amide H C BEH HILIC.0 H CH 0 6 8 0 mobile phase ph Relative Retention to Acenaphthene (k').0.0.0 H methacrylic acid BEH Amide BEH HILIC 0 6 8 0 mobile phase ph 009 ihon Waters K.K. 9 : ph tion to e (k') Relative Retent Acenaphthene Relative Retention to Acenaphthene (k') 6.0 0.0.0.0.5.0.5.0 0.5 H H cytosine BEH Amide BEH HILIC 0 6 8 0 mobile phase ph H CH nortriptyline BEH Amide BEH HILIC 0 6 8 0 mobile phase ph ion to e (k') Relative Retenti Acenaphthene Relative Retention to Acenaphthene (k') H 6.0 0.0 H.0 8.0 6.0.0.0 H H H allantoin norepinephrine BEH Amide BEH HILIC 0 6 8 0 mobile phase ph H H H BEH Amide BEH HILIC 0 6 8 0 mobile phase ph 009 ihon Waters K.K. 0 0
: ph ph ph * *.Gara J.E. Wyndham K.D. J. Liq. Chromatogr. Relat. Technol. 006 9 05-05 009 ihon Waters K.K. : vs. HILIC XBridge TM HILIC Atlantis HILIC Silica Injection 0 Injection 0 0 Injection 000 Injection 800 0 Injection 000 Injection 000 0 0 0.60 0.80.00.0.0.60.80.00 0 0.60 0.80.00.0.0.60.80.00 Column:. x 50 mm.5 µm Mobile Phase A: 95:5 acetonitrile: water with 0 mm H + CH C- ph 5.5 ph 5.5 000 Mobile Phase B: 50:50 acetonitrile: water with 0 mm H + CH C- ph 5.5 Flow Rate: 0.5 ml/min Gradient: 99%B over minutes %B at. min hold for 0.min Injection Volume:.0 µl (Full loop) Temperature: 0 o C Detection: UV @ 5 nm : uracil : 5-fluorouracil : cytosine 009 ihon Waters K.K.
: 0mM 0.% / 50:50 BEH Amide / 60:0 50 50 0 0 5-8 5-8 009 ihon Waters K.K. : 95-50% 5% ( ) ) 95% : 0% in : 9: / / / ( ) ) (75%) 009 ihon Waters K.K.
( ) 75% HILIC 75:5 : 009 ihon Waters K.K. 5 : 0.60 0.50 0.0 0 00% H S 0 0.50.00.50.00.50.00 0.60 50 AC: H 0.50 0.0 0 S 0 0.50.00.50.00.50.00 0.60 0.50 0.0 75 AC: 5 H S ACQUITY UPLC BEH HILIC. x 00 mm.7 µm Analytes. Methacrylic acid. Cytosine. ortriptyline. icotinic acid 0 0 0.50.00.50.00.50.00 009 ihon Waters K.K. 6
: 0.60 0.50 0.0 00 0 S 00% MeH 0 0.50.00.50.00.50.00 0.60 50 AC: 50 MeH 0.50 0.0 0 000 S ACQUITY UPLC BEH HILIC. x 00 mm.7 µm Analytes. Methacrylic acid. Cytosine. ortriptyline. icotinic acid 0 0.50.00.50.00.50.00 0.60 0.50 0.0 S 75 AC: 5 MeH 0 0 0.50.00.50.00.50.00 009 ihon Waters K.K. 7 MS : vs. HILIC Intens ity.0x0 7.5x0 7 0x0.0x0 7.5x0 7 XBridge TM C 8 00% H H C + CH H C CH. Acetylcholine (Ach).0x0 7 5.0x0 6 H C + H C CH H intensity.0x0 7 0.5 X response.5x0 7.0x0 7.5x0 7.0x0 7 5.0x0 6 8.6 X response HILIC XBridge TM HILIC 8 AC:.5 MeH:.5 H. Choline (Ch) HILIC MS 0.60 0.80.00.0.0.60.80.00 009 ihon Waters K.K. 8
MS ph ph vs HILIC inte ensity x0 6 x0 6 ph intensity 0 x0 6 x0 6 ph Morphine 0 x0 6 HILIC HILIC : intensity x0 6 ph. ph. 0 0.50.00.50.00.50.00.50.00.50 5.00 009 ihon Waters K.K. 9 SPE SPE / SPE * HILIC *Jernal M. Teitz D. uyang Z. J.Chromatogr. B 7 (999) 50. 009 ihon Waters K.K. 0 5
: SPE SPE XBridge TM HILIC asis MCX µelution Plate / / 00 µl MeH/ 00 µl H 0 ng/ml 6-acetylmorphine spiked into human plasma 00 µl spiked human plasma with % H P 00 µl % HCH in H 00 µl MeH 00 µl AC 0 ng/ml morphine spiked into human plasma 50 µl 5% H H in AC / 0.00.00.00.00 5.00 6.00 009 ihon Waters K.K. Sub-µm : HILIC Sub- µm 0 µm Atlantis HILIC Silica H.7 µm ACQUITY UPLC BEH HILIC H (µm) 0 H Cytosine (B) Conditions 0 ACQUITY UPLC with TUV. x 50 mm column 90% AC with 0 mm ammonium formate (pre-mixed) Inj. =.0 µl Full loop injection Test Probe: Cytosine (k =.5) 5 nm 0 WW 90% AC with 0 mm ammonium formate 0 Hz, Tc = 0 5 linear velocity (mm/sec) 009 ihon Waters K.K. 6
UPLC : intensity 5.0x0 7.5x0 7.0x0 7.5x0 7 0.0x0 7.5x0 7.0x0 7 V 0 = 0.5 min.5x0 7.0x0 7 5.0x0 6 HPLC XBridge TM HILIC.5 µm F = 0.5 ml/min H C + CH H C CH. Acetylcholine (Ach) H C + H H C CH intensity 5.0x0 7.5x0 7 0x0.0x0 7.5x0 7.0x0 7.5x0 7.0x0 7 V 0 = 0.8 min.5x0 7.0x0 7 5.0x0 6 UPLC ACQUITY UPLC BEH HILIC.7 µm F =.0 ml/min.6x Higher Response Ach.6X Higher Response Ch X Faster at Scaled Flow Rate 0.60 0.80.00.0.0.60.80.00. Choline (Ch) UPLC Technology HPLC 009 ihon Waters K.K. ACQUITY UPLC BEH Amide UPLC /MS ph 00 ACQUITY UPLC BEH Amide. Maleic acid ( ppm). Pyruvic acid (50 ppm) HILIC Conditions ph 9.0 SIR Mode Electrospray -. Lactic acid (50 ppm). Succinic acid (50 ppm) 5. Fumaric acid (50 ppm) % 5 0 0 0.80.0.60.00.0 009 ihon Waters K.K. 7
ACQUITY UPLC HSS T Atlantis T ACQUITY UPLC BEH C 8 XBridge TM C 8 ph ph MS ACQUITY UPLC BEH HILIC ACQUITY UPLC BEH Amide XBridge TM HILIC Atlantis HILIC Silica LC/MS SPE HILIC Amide 009 ihon Waters K.K. 5 8