Agilent Bond Elut Plexa PCX Cation Exchange SPE handbook
Contents
1. connection 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 2011 Printed in the USA July 15 2011 5990 8400EN 7 Agilent Technologies2. 18 2 1 x 5 0 mm 2 7 um p n 699775 902 LC MS Agilent 1260 Infinity LC MS A 0 1 formic acid in H 0 B 0 1 formic acid in MeOH Flow rate 0 4 mL min Injection volume 10 uL Gradient Time min B 0 10 40 90 4 1 10 6 5 10 Temperature sample 25 C column ambient lon source ESI with JetStream Gas temperatue 350 C Gas flow 10 L min Nebulizer 35 psi Sheath gas temperature 400 C Sheath gas flow 12 L min Capillary 4000 V LC MS MS wi Injection of blank plasma Syringe pump continuous infusion of drug mixture 50 ng mL at 20 pL min Figure 1 Schematic of ion suppression comparison experiment setup 1 For calibration and recovery plasma was spiked with drug compounds of corresponding concentrations For ion suppression comparison blank plasma samples were processed with SPE Table 1 Samples Results and Discussion MS MS Collision Good separation and retention among all analytes were pha ng transition energy Fragmentor achieved and shown in Figure 2 Chromatograms shown in Acebutolol 9 40 171 337 2 gt 116 1 20 128 Figure 3 were obtained during continuous infusion of drug Ranitidine 8 20 0 27 315 2 176 1 12 92 mixture with blank plasma sample injections processed by Nadolol 967 081 310222541 12 92 each SPE product The data show clearly that Agilent Bond Kenatol aki iie oe ee D Elut Plexa PCX has reduced ion suppression when compared to its compe
3. 85 90 95 100 105 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 i Concentration ng mL Concentration ng mL Figure 4 Calibration curves of six beta blockers at nine concentration levels 0 01 0 05 0 1 0 5 1 5 10 50 and 100 ng mL Table 2 Agilent Bond Elut Plexa PCX Data Summary 5 ng mL 50 ng mL 100 ng mL Correlation pKa log P LOD ng mL LOQ ng mL Recovery RSD Recovery RSD Recovery RSD coefficient R Atenolol 9 60 0 16 0 05 0 1 109 0 1 2 95 6 2 3 95 5 3 3 0 997 Nadolol 9 67 0 81 0 01 0 05 110 8 14 120 7 1 5 95 4 1 6 0 998 Acebutolol 9 40 1 71 0 01 0 1 113 9 0 9 108 6 2 0 98 7 2 4 0 999 Propranolol 9 42 3 48 0 05 0 1 120 2 1 1 103 5 2 7 93 6 2 5 0 999 Procainamide 9 32 0 88 0 05 0 1 93 0 2 1 104 5 1 8 96 9 3 9 1 Ranitidine 8 20 0 27 0 05 0 1 90 7 1 9 96 4 2 7 91 1 3 9 1 Conclusion Agilent Bond Elut Plexa PCX showed reduced ion suppres sion when compared to their competitive SPE products Low LOD 0 01 0 05 ng mL and LOQ 0 05 0 5 ng mL were obtained resulted from minimized ion suppression Excellent correlation coefficients R gt 0 995 and good recovery data were obtained with very good RSD as well For More Information These data represent typical results 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
4. e oO e e e e Author Mike Chang Agilent Technologies Inc 25200 Commercentre Drive Lake Forest CA 92630 USA Agilent Bond Elut Plexa PCX Cation Exchange SPE A Destination to a Better Sensitivity in LC MS Bioanalysis Resulting from Minimized lon Suppression Application Note BioPharma Introduction Throughout the drug development process in pharmaceutical industry it is of essence to develop and validate fast methods for bioanalysis without losing sensitivity lon suppression often can be the most commonly encountered issue in achieving that goal which causes low recovery inaccuracy as well as increased instrument maintenance cost and time While ion suppression cannot be fully avoided when biological samples are handled it should be avoided as much as possible The nature of hydroxylated surface on Agilent Bond Elut Plexa PCX makes it stand out among other cation exchange SPE products with amide residue on the surface of the sorbent The presence of amide residue causes increased interac tion between the SPE sorbent and the endogenous material in biological sample which can be directly responsible for ion suppression during bioanalysis Due to hydroxylation of the sorbent s surface Bond Elut Plexa PCX reduces the interac tion between the sorbent and the endogenous material in the biological matrices hence they achieve improved sensitivity The following experiment shows clear evidence of ion suppression
5. reduction and improved sensitivity with Bond Elut Plexa PCX mono dispersed polymeric SPE oe Agilent Technologies Materials and Methods SPE reagents and solutions 2 H PO Add 20 pL H PO to 1 mL H 0 2 formic acid Add 20 uL formic acid to 1 mL H 0 MeOH Reagent grade or better 50 50 MeOH ACN Add 1 mL MeOH to 1 mL ACN 5 ammonia in 50 50 MeOH ACN Add 50 uL diluted NH OH to 1 mL 50 50 MeOH ACN SPE Method All samples were processed by the same SPE method SPE products Agilent Bond Elut Plexa PCX 96 well plate 10 mg p n A4968010 Competitor W 96 well plate 10 mg Competitor P 96 well plate 10 mg Sample 100 pL human plasma Pretreatment Dilute with 300 uL 2 H PO Conditions 1 500 pL MeOH 2 500 uL H 0 Load 400 uL diluted sample from pretreatment actual plasma amount 100 pL Wash 1 500 uL 2 formic acid 2 500 uL 50 50 ACN MeOH Elute 2 x 250 pL 5 ammonia in 50 50 ACN MeOH YF i a i mixer splitter e mE a Experiment Design For ion suppression comparison drug compound mixture 50 ng mL was continuously infused by a syringe pump at 20 L min while a blank plasma sample was injected Blank plasma samples were prepared by Agilent Bond Elut Plexa PCX and two competitor s products based on the SPE methods specified in the previous section MS transition 184 gt 184 was selected for lipid contents monitoring during the analysis LC Conditions Column Agilent Poroshell 120 EC C
6. s x10 y 0 380413 x xo Jy 0 117117 x 1 24 m 36 R 0 99887629 12 1R 0 99954504 32 104 o 3 0 0 94 228 2 a 6 26 Acebutolol 608 Ranitidine a24 Q 222 anI 20 ggd 218 g B16 B05 S 312 g 04 1 0 0 34 08 0 24 0 4 0 1 0 2 0 0 0 0 0 2 0 1 5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 Concentration ng mL Concentration ng mL ai nadolol 9 levels 9 levels used 9 points 9 points used 0 OCs atenolol 9 levels 9 levels used 9 points 9 points used 0 QCs 424y 0 398220 x 70 y 0 066168 x 38 R 0 99817817 C R 0 99661424 3 64 04 344 5 57 3 24 3 04 o 5 0 2284 500 Nadolol g Atenolol 2374 24 0 22 S35 gis Q30 18 B25 2 12 2205 08 154 064 104 o44 024 0 55 0 04 0 0 0 2 0 5 me 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 Concentration ng mL Concentration ng mL propanolol 9 levels 9 levels used 9 points 9 points used 0 OCs procainamide 9 levels 9 levels used 9 points 9 points used 0 OCs x10 Jy 0 131686 x 0 y 0 346187 x 137R 0 99895038 3 44R 0 99975257 124 324 114 al 2104 2 261 gos Propranolol S73 Procainamide 0 8 zPY 307 Sial Sos Lisi B05 amp ia 04 1 04 0 3 0 84 0 24 064 0 44 01 0 25 0 0 0 0 01 0 2 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80
7. titive SPE products Propranolol 9 42 3 48 260 2 gt 116 2 16 92 Procainamide 9 32 0 88 236 2 gt 120 1 16 92 Excellent limit of detection LOD and limit of quantitation Metoprolol 970 190 26821162 16 92 LOQ were achieved with Bond Elut Plexa PCX A recovery ISTD experiment was performed at three different concentration levels low mid and high n 6 and the data are shown in Table 1 with excellent recovery and RSD All compounds showed good linearity with correlation coefficients R gt 0 995 Figure 4 x10 64 5 4 3 2 Acebutolol x10 1 0 0 8 0 6 i Ranitidine x10 4 34 2 1 Nadolol x10 1 0 0 8 0 6 s Atenolol x10 2 0 15 1 0 05 Propranolol x10 4 34 2 Boks 1 Procainamide 01 03 05 07 09 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 Counts vs aquisition time min Figure 2 MS chromatogram of spiked plasma sample processed by Agilent Bond Elut Plexa PCX 5 ng mL each 2 2 A Agilent Bond Elut Plexa PCX an N Competitor W Competitor P 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 45 5 0 5 5 6 0 Counts vs aquisition time min Figure 3 Lipid contents monitoring of blank plasma sample injection by 184 gt 184 m z transition acebutolol 9 levels 9 levels used 9 points 9 points used 0 QCs ranitidine 9 levels 9 levels used 9 points 9 points used 0 QC
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Agilent Bond Elut Plexa PCX Cation Exchange SPE handbook