Agilent Technologies Hydrophilic Interaction Chromatography (HILIC) Separation of Basic Drugs using MS/MS Detection Application Note
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1. mM HCOONH in water B 8 mM HCOONH in 95 acetonitrile MeCN 5 water Gradient 5 B to 90 B in 10 min Column temperature 40 C Sample volume 5 ul Flow rate 0 3 mL min HILIC Everything same as for RPLC except for column and gradient conditions Column ZORBAX RX SIL 2 1 mm x 150 mm 5 pm Gradient 100 B to 50 B in 10 min Mass Spectrometry Instrument Series 1100 LC MSD Trap lonization Positive ESI Scan range 100 500 m z SIM ions m z 315 330 Drying gas 10 L min at 350 C Nebulizer gas 45 psi Fragmentor voltage 0 25 V Results and Discussion In order to select the best SIM for online monitor ing the MS spectra of the drug standards were run Figure 2 shows that an M 1 ion was observed but ions at m z of 192 0 and 175 9 were used for paroxetine and ranitidine respectively since they showed stronger signals These ions were monitored in subsequent runs J 192 0 Paroxetine a MW 329 4 150 9 M 1 7 330 2 5 109 1 123 0 175 0 208 0 313 1 if alk jal al L l 1 aaa et L r 7 1 100 150 200 250 300 350 400 450 m z 175 9 124 0 E Ranitidine 4 MW 314 4 1 M 1 315 1 4 224 0 270 0 ARO ggl ye tL ok 241 0 fat ly hdl ee oe a a m 100 150 200 250 300 350 400 450 m z Figure 2 MS MS spectra of drug standards showing ions selected for subsequent fragmentation The RPLC gradient elution total ion chromatogram of standards shown in Figure 3 gave an2. measured at the 100 ppb level serum Recov ery was 79 for paroxetine and 98 for ranitidine both acceptable at these levels ZORBAX RX SIL ete aad 100 ppb Serum MeCN 1 3 100 ppb each spiked 5 uL injection Ranitidine 100 ppb m z 315 Abundance 0 2 4 6 Time min Ranitidine 100 ppb m z 315 176 Recovery 98 Paroxetine 100 ppb m z 330 192 Recovery 79 Abundance LC MS MS 0 2 4 6 Time min LC MS and LC MS MS analysis of paroxetine and ranitidine spiked into human serum with ZORBAX RX SIL Column Figure 8 Conclusion Ranitidine and paroxetine were successfully sepa rated using the HILIC mode The elution order was reversed compared to the RPLC mode Good lin earity was shown over the 0 5 100 ppb levels A spiked serum was cleaned up by protein precipita tion and an aliquot was directly injected into the HILIC ESI MS system Recovery was found to be 98 for ranitidine and 79 for paroxetine at the 100 ppb level References 1 M Przybyciel and R E Majors 2002 LCGC No America 20 6 516 523 2 R E Majors and M Przybyciel 2002 LCGC No America 20 7 584 593 3 W Naidong 2003 J Chromatogr B 796 209 224 For More Information For more information on our products and services visit our Web site at www agilent com chem www agilent com chem Agilent shall not be liable for errors contained herein or for incidental or consequential damages in co
3. Hydrophilic Interaction Chromatography HILIC Separation of Basic Drugs using o o pa e Application one 0 e a e o e Drug Analysis es e s Authors Tatsunari Yoshida Kazuo Yamanaka and Hiroki Kumagai Yokogawa Analytical Systems Inc 9 1 Takakura Cho Hachioji Shi Tokyo 192 0033 Japan Ronald E Majors Agilent Technologies Inc 2850 Centerville Road Wilmington DE 19808 1610 USA Abstract The basic drugs ranitidine and paroxetine were success fully separated using hydrophilic interaction liquid chro matography HILIC The elution order was reversed compared to reversed phase liquid chromatography RPLC Good linearity was shown over the 0 5 100 ppb levels A spiked serum was cleaned up by protein precipi tation and an aliquot was directly injected into the HILIC ESI MS system Recovery was found to be 98 for ranitidine and 79 for paroxetine at the 100 ppb level Introduction Because of its versatility RPLC is the most widely used technique in all of HPLC It separates mole cules based on the hydrophobic interactions between the nonpolar stationary phase and the MS MS Detection organic portions of typical analytes However the retention of polar analytes often requires a highly aqueous mobile phase to achieve retention Highly aqueous systems sometimes lead to problems such as phase collapse dewetting 1 decreased sensi tivity in mass spectroscopic detection due to poor mob
4. ed for both drugs 1 0E 047 HILIC separation ZORBAX RX SIL Silica column 2 1 mm x 150 mm 0 5 ppb Paroxetine Ranitidine B95 B50 10 min gradient m z 330 192 m z 315 176 a 0 0E 00 4 0 2 4 6 8 10 12 Time min Figure 5 LC MS MS Separation of paroxetine and ranitidine on ZORBAX RX SIL column HILIC mode 0 5 ppb level We constructed a calibration curve as shown in Figure 6 and both drug compounds gave good lin earity with an acceptable correlation coefficient over the expected concentration range 1 0E 07 Paroxetine 0 5 100 ppb r 0 999 Ranitidine Abundance 0 0E 00 120 ppb Figure 6 Linearity of paroxetine and ranitidine on ZORBAX RX SIL Column HILIC Mode Next a human serum sample was spiked with a 5 uL aliquot of each drug compound 20 ppm and the sample was prepared according to the sample preparation protocol outlined in Figure 7 Control serum 245 uL Spiked with 5 uL 20 ppm paroxetine and ranitidine solution Acetonitrile 750 uL was added to precipitate proteins Centrifugation at 12000 rpm 8 min Solution phase 5 pL was directly injected into HPLC Figure 7 Sample preparation procedure of spiked serum A 5 uL aliquot of the cleaned up serum sample containing the drugs was injected into the HILIC column The resulting ion chromatograms of Figure 8 upper trace LC MS and lower trace LC MS MS show that both compounds were able to be
5. excellent and relatively fast separation of the two drugs However with ranitidine there was some tailing at the 100 ppb parts per billion level Since we were interested in the measurement of lower levels in serum this separation was deemed unacceptable so we switched our attention to the HILIC conditions Paroxetine m z 330 192 RPLC separation ZORBAX Eclipse XDB C18 2 1 mm x 150 mm 100 ppb B5 B90 10 min gradient Abundance Ranitidine m z 315 176 0 2 4 6 8 10 12 Time min Figure 3 LC MS MS separation of paroxetine and ranitidine on ZORBAX Eclipse XDB C18 column RPLC mode Figure 4 depicts the separation of the two drug standards using HILIC on a silica gel column Note that the elution order was reversed as might be expected but the peak shape for ranitidine was improved over the RPLC separation The selectivity was not as good as with RPLC nevertheless excel lent baseline resolution was achieved In addition under the conditions employed the separation was faster than with RPLC HILIC separation Paroxetine ZORBAX RX SIL Silica column 2 1 mm x 150 mm m z 330 192 100 ppb B95 B50 10 min gradient S G zi lt Ranitidine m z 315 176 0 2 4 6 8 10 12 Time min Figure 4 LC MS MS Separation of paroxetine and ranitidine on ZORBAX RX SIL column HILIC mode 100 ppb level Figure 5 shows the same separation but now at the 0 5 ppb level Good sensitivity was not
6. ile phase desolvation and ion suppression and still may not allow retention of very polar analytes Some specialized packings were developed to allow the use of highly aqueous systems such as polar embedded phases hydrophilically endcapped reversed phase bonded silicas wide pore low density bonded silicas short chain phases and other special designs 2 An alternative technique for the separation of highly polar analytes that gets around some of the problems associated with RPLC is HILIC HILIC requires a high percentage of a nonpolar mobile phase and a polar stationary phase similar to the requirements in normal phase chromatography NPC However unlike NPC which uses nonpolar solvents such as hexane and methylene chloride and tries to exclude water from the mobile phase HILIC requires some water in the mobile phase to maintain a stagnant enriched water layer on the surface into which analytes may selectively parti tion In addition water miscible organic solvents are used instead of the water immiscible solvents used in NPC With HILIC sorbents such as bare silica bonded diol and polyhdroxyethylaspar tamide are used Polar analytes are well retained a Agilent Technologies and elute in order of increasing hydrophilicity just the inverse of RPLC Sometimes under HILIC con ditions polar analytes will show a very different selectivity compared to RPLC It was demonstrated recently that the mechanism of HILIC involve
7. nnection with the furnishing performance or use of this material Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies Inc 2005 Printed in the USA September 8 2005 5989 3761EN z Agilent Technologies
8. s var ious combinations of hydrophilic interaction ion exchange and reversed phase retention by the siloxane on the silica surface 3 Basic drugs with amine functionality are often sep arated by RPLC and under acidic conditions when protonated may show decreased retention In the present study we investigated the separation of basic drugs in serum using RPLC on a C18 column and HILIC on bare silica gel employing electrospray ionization ESI and MS MS detection Basic Drugs Studied The structures of the basic drugs studied are depicted in Figure 1 Paroxetine Figure la is a psychotropic drug that is administered orally It has a water solubility of 5 4 mg mL and in its free base form its molecular weight MW is 329 36 Ranitidine Figure 1b is an antiulcerative and works by decreasing the amount of acid that the stomach produces It is freely soluble in water and methanol but sparingly soluble in ethanol Its MW is 314 41 in its free base form F N a Paroxetine 0 Antidepressant MW 329 36 0A b Ranitidine 0 NSAN NS Antiulcerative Xy TL MW 314 41 7 NO Figure 1 Structures of drug compounds studied Chromatographic System Since mass spectrometry MS using ESI was employed for detection we elected to use 2 1 mm internal diameter id columns where the normal flow rate was more compatible RPLC Instrument Agilent Series 1100LC Column ZORBAX Eclipse XDB C18 2 1 mm x 150 mm 5 pm Mobile phase A 8
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