High Productivity Maureen Joseph Columns with LC/MS Anne E. Mack Applications- William E. Barber Integrating Fast LC with MS Workshop Agilent Technologies Pittcon 2011

March 15, 2011

Confidentiality Label 1 March 28, 2011 Outline

Column choices for productivity and fast LC • Smaller particle size columns MS specific concerns • Particle size benefits for MS analyses – Sample type does matter • Detector speed • Separation quality vs. compatibility with the MS • Reliability

Confidentiality Label 2 March 28, 2011 Smaller Particle Size Column Choices Key for Improving Productivity in LC Totally porous particle columns • Small particle sizes – including sub 2um Superficially porous particle columns • Mainly available in 2.6-2.7um

“I have been truly amazed/impressed by the performance of the small particle columns!! The reproducibility is incredible and so is the performance (peak and loading capacity). We did 110 injections of a non-depleted plasma digest spiked with heavy labeled proteins and the RT reproducibility is incredible!”

Confidentiality Label 3 March 28, 2011 How Do These Choices Improve Productivity in LC and LC/MS Separations?

More resolution • Increased resolution of samples • Closely eluting peaks resolved with the added efficiency • More compounds resolved in one analysis, reducing number of analyses needed for the same results • Improving quality of results – accuracy of integration or identification with LC/MS More speed • Faster methods mean more samples analyzed in limited amount of time • Speed, without compromising resolution

Confidentiality Label 4 March 28, 2011 What are the Primary Questions and Concerns when Using these Columns with MS or MS/MS

Are the benefits the same as with UV detection?

• Do you need the smaller particle size or is 3-3.5um good enough? Is the chromatography the same and are there limitations when using these columns with MS? • Does choosing an MS compatible mobile phase compromise results? Are there advantages and disadvantages to small particle size columns particular to MS • Same molecular weight analytes

Confidentiality Label 5 March 28, 2011 Are the Benefits the Same? High Efficiency/High Resolution for Fast Analysis

Gradient separation – comparison of peak capacity

Conditional Peak Capacity Equation Analgesic Compounds in Analysis High Resolution/High Peak Capacity Needed Compounds (in elution order) with Identifying Mass: acetaminophen: m/z 109 Two sets of compounds have the caffeine: m/z 194 same molecular weight. 2-acetamidophenol: m/z 109 Therefore chromatographic resolution acetanilide: m/z 135 is required to identify the compounds. acetylsalicylic acid: m/z 120 phenacetin: m/z 179 salicylic acid: m/z 120 sulindac: m/z 356 piroxicam: m/z 332 tolmetin: m/z 257 ketoprofen: m/z 254 diflunisal: m/z 332 diclofenac: m/z 295 celecoxib: m/z 381 ibuprofen: m/z 160

Confidentiality Label 7 March 28, 2011 Analysis of 15 Analgesic Compounds on 3.5um, 1.8um Totally Porous and 2.7um Superficially Porous Columns

ZORBAX Eclipse Plus C18, 3 x 100 mm, 3.5 µm Same method can be used Ibuprofen: with all 3 columns due PW =0.014 1/2 to similar selectivity S/N=182 n =43 c 1.8 & 2.7 µm columns: • taller, narrower peaks ZORBAX RRHD Eclipse Plus C18, 3 x 100 mm, 1.8 µm • >40% more sensitivity, Ibuprofen: as noted by the S/N of PW1/2=0.012 ibuprofen S/N=353 • Conditional peak n =54 c capacity >20% higher than the 3.5 µm column Poroshell 120 EC-C18, 3 x 100 mm, 2.7 µm • 15 compounds, 2 min Ibuprofen:

PW1/2=0.012 S/N=256

nc=56 2 min Shorter columns with smaller particles allows faster analyses, maintains resolution of 15 analgesic compounds

ZORBAX Eclipse Plus C18, Average Conditional peak capacity 3 x 100 mm, 3.5 µm PW =0.025 same for: 50 mm 1.8 & 1 mL/min 1/2 nc=43 2.7 µm columns and 100 mm 3.5 µm column in half the 2 min analysis time, ZORBAX Eclipse Plus C18, 3 x 50 mm, 1.8 m µ Average 100-mm gradient: 1 mL/min PW =0.0092 1/2 15-95% CH3CN in 1.5 min nc=39 Stop Time: 2 min

1 min 50-mm gradient: Poroshell 120 EC-C18, 15-95% CH3CN in 0.75 3 x 50 mm, 2.7 µm Average min 1 mL/min Stop Time: 1.05 min PW1/2=0.0086

nc=43

1 min For fastest results: 1.8-µm RRHD columns are used for 15 component analgesic mixture

ZORBAX Eclipse Plus C18, Average Results: • Loss of some peak capacity 3 x 100 mm, 3.5 µm PW =0.025 1 mL/min 1/2 on 50mm long column. nc=43 • Shorter column, higher flow rate provides 80% faster analysis. • Good result on 1.8um column in comparison to longer 3.5um column. 1.9 min• High flow rate possible due to 1200 bar pressure limit. Agilent ZORBAX RRHD Eclipse Plus C18, Average 3 x 50 mm, 1.8 µm • 100-mm gradient: PW1/2=0.0051 2.75 mL/min • 15-95% CH3CN in 1.5 min nc=26 • Stop Time: 2 min

• 50-mm gradient:

• 15-95% CH3CN in 0.27 min • Stop Time: 0.41 min

80% Faster Analysis 0.38 min Good peak integration and quantification with MS when all analytes have unique masses - Analysis of 15 analgesics

Agilent ZORBAX RRHD Eclipse Plus C18, 3 x 50 mm, 1.8 µm 2.75 mL/min, 15-95% CH3CN in 0.27 min, Stop Time: 0.41 min The total ion chromatogram TIC Scan (TIC) shows piroxicam tolmetin coeluting with tolmetin

Extracted ion chromatograms (EIC) of each mass fragment allow isolation of EIC overlay of all 15 individual compounds compounds Viewing EIC’s separately allows for simple integration and Individual EIC of m/z 257 (tolmetin) quantification of any compound, as shown here with tolmetin

Note: EIC’s cannot separate coeluting peaks when they are identified by the same mass fragment (ex. Isomers) High Resolution of Peptide Mixture for Peptide Quantitation on 1.8um Column Zoom view Column: Eclipse Plus C18, 2.1 x 150mm, 1.8um

92 peptides spiked into digested plasma or digested depleted plasma (n=3 of each) Solvent: A=0.1% formic acid in water; B=0.1% formic acid in 90% ACN/water Gradient: min %B 0 3 2 6 35 20 40 25 45 45 46 90 50 90 50.1 3

In addition to excellent resolution, excellent reproducibility was also obtained.

These samples were kindly provided by Derek Smith and Christoph H. Borchers from the UVic-Genome BC Proteomics Centre

Confidentiality Label 12 March 28, 2011 Reducing Analysis Time for Peptide Quantitation: Eclipse Plus C18, 1.8 µm, 2.1 x 150 mm Column

Original 20 minute method

New 9 minute method

These samples were kindly provided by Leigh Anderson and Terry Pearson, Anderson Forschung Group A 1 minute Analysis by LC/MS –

Sample: 3 µL of 3.3 µg/mL each of , gallocatechin, epigallocatechin, , caffeine, epicatechin, , , epicatechin gallate, catechin gallate in H O/CH CN 2 3 Agilent Poroshell 120 SB-C18, 2.1 x 50 mm, 2.7 µm 1.00 mL/min 1 425 bar

0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 1.05 1.1 1.15 1.2 1.25 1.3 1.35 1.4 1.45

1 1.25 mL/min A: 0.2% CH3COOH in H2O 505 bar B: CH3CN 40 oC Column: Agilent Poroshell 120 SB-C18, 2.1 x 50 mm, 2.7 µm Source: 350 oC, 10 L/min, 50 psi, -/+3500 V Acquisition: SIM- (169, 305, 289, 457, 441), SIM+ (195) Sample: 1.5 µL injection of 0.003 mg/mL each of GA, GC, EGC, C, Caf, EC, EGCG, GCG, ECG, CG in 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 1.05 1.1 1.15 H2O/CH3CN

1 1.00 mL/min: 1.50 mL/min Time (min) 0.00 0.50 1.00 585 bar %B 10 15 27 1.25 mL/min: Time (min) 0.00 0.42 0.83 1.0 min %B 10 15 27 1.50 mL/min: Time (min) 0.00 0.36 0.71 %B 10 15 27 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 Antidepressants and Other Compounds in Blood – by LC/MS Very dirty sample – what is the impact of this sample matrix? Will the matrix compounds interfere with the analytes of interest? What type of analysis time can be achieved with a small particle column?

Confidentiality Label 15 March 28, 2011 LC/MS/MS Chromatograms of 100 ng/mL sample LC/MS/MS Chromatograms of Sample Compounds Poroshell 120 EC-C18, 2.1 x 100mm, 2.7um

x10 3 +ESI MRM Frag=112.0V [email protected] (278.2000 -> 117.0000) HQC_6.d 5.1 1 1 5 4.9 4.8 Fast Analysis – Adequate LC/MS/MS Resolution 4.7 4.6 4.5 All compounds identified 4.4 4.3 4.2 4.1 4 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3 2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 4.4 4.5 4.6 Counts vs. Acquisition Time (min) 4.8 min Whole Blood Extract – Background Scan

x10 7 +ESI TIC Scan Frag=80.0V AOAC_BLANK_110228b.d 1.375 1 1 1.35 1.325 1.3 1.275 1.25 Poroshell 120 EC-C18, 2.7 µm 1.225 1.2 2.1 x 100 mm, PN 695775-902 1.175 1.15 Mobile Phase A: 5 mM Ammonium 1.125 1.1 acetate, pH 5; 20:80 MeOH:Water 1.075 1.05 1.025 B: 5 mM Ammonium Acetate in 1 0.975 ACN 0.95 0.925 GT (ºC): 300 0.9 0.875 GF (l/min): 7 0.85 0.825 Nebulizer (psi): 40 0.8 0.775 SGT (ºC): 400 0.75 0.725 SFG (l/min): 12 0.7 0.675 Capillary (V): 3500 0.65 0.625 0.6 NV (V): 500 0.575 0.55 0.525 0.5 0.475 0.45 0.425 0.4 0.375 0.35 0.325 0.3 0.275 0.25 0.225 0.2 0.175 0.15 0.125

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 Counts vs. Acquisition Time (min) QuEChERS Extraction of Whole Blood MS Scan mass range 100-800, scan time 20 sec, Fragmentor 80 V, positive polarity. Injection volume 10 µL, 20% B to 75% B over 5.5 minutes. Hold 75% B for 2 minutes. Extract of Whole Blood – Sample and Background Poroshell 120 Provides the Resolution

x10 7 +ESI TIC Scan Frag=80.0V ORG_250NG_IS_2.d 1.375 1 1 1.35 1.325 1.3 1.275 1.25 1.225 1.2 1.175 1.15 1.125 1.1 1.075 1.05 1.025 1 0.975 0.95 0.925 0.9 0.875 0.85 0.825 0.8 0.775 0.75 0.725 0.7 0.675 0.65 0.625 0.6 0.575 0.55 0.525 0.5 0.475 0.45 0.425 0.4 0.375 0.35 0.325 0.3 0.275 0.25 0.225 0.2 0.175 0.15 0.125 0.1 0.075 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 Counts vs. Acquisition Time (min) QuEChERS Extraction of Whole Blood overlay 250 ng/mL 10 compounds; MS Scan mass range 100-800, scan time 20 sec, Fragmentor 80 V, positive polarity LC/MS/MS Chromatograms After Quechers Extract Poroshell 120 EC-C18, 2.1 x 100mm, 2.7um

x10 3 +ESI MRM Frag=112.0V [email protected] (278.2000 -> 117.0000) HQC_6.d 5.1 1 1 5 4.9 4.8 Fast Analysis – Adequate LC/MS/MS Resolution 4.7 4.6 4.5 All compounds identified 4.4 4.3 4.2 LC/MS/MS Chromatograms of 100 ng/mL spiked whole blood sample after mini- 4.1 4 QuEChERS extraction; AOAC (NaAc) and d-SPE (PSA) 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3 2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 4.4 4.5 4.6 Counts vs. Acquisition Time (min) 4.8 min Are there more advantages to smaller particle size columns for LC/MS? Experiment: Compare results on columns with different particle sizes. • Sample 1 – Toxicology sample analyzed by LC/MS/MS • Sample 2 – Analgesics sample analyzed by LC/MS Observations: • peak width – sharper? • peak height – taller? • sensitivity - S/N • peak capacity – greater? • Analysis time – shorter? • Resolution – where do we need how much?

Confidentiality Label 20 March 28, 2011 Sub 3um Columns Produce Sharp, Efficient Peaks for LC/MS/MS – Toxicology Sample

Agilent Poroshell 120 EC-C18, 2.1 x 100 mm, 2.7 µm 1.8-µm particle column: tallest, sharpest peaks, 2.7-µm particle column Agilent ZORBAX RRHT Eclipse Plus C18, 2.1 x 100 mm, has similar results 1.8 µm 3.5-µm column produces slightly shorter peaks, 5-µm column generates Agilent ZORBAX Eclipse Plus C18, 2.1 x 100 mm, 3.5 µm significantly broader, shorter peaks

Agilent ZORBAX Eclipse Plus C18, 2.1 x 100 mm, 5 µm Conditions

A: 5mM ammonium formate w/ 0.01% formic acid (1L water + 0.3153g ammonium formate + 0.1mL formic acid)

B: acetonitrile w/ 0.01% formic acid (1L acetonitrile + 0.1mL formic acid)

0.5mL/min; 10%B at t0, ramp to 15%B in 0.5min, ramp to 50%B in 2.5min, ramp to 95%B in 1min, hold 95%B for 2min; stop time 6min, post run 2min

Sample: injector program: draw 5µL water, draw 1µL LC/MS Toxicology Test Mixture (PN 5190-0470), diluted 1:10, inject

TCC=60oC

RRHT Eclipse Plus C18, 2.1 x 100 mm, 1.8 um

MS Source: electrospray AP-ESI, drying gas temperature and flow: 350oC, 12L/min, nebulizer gas pressure: 30psi, capillary voltage: 2000V

MS Acquisition: dynamic MRM, positive ionization polarity

Confidentiality Label 22 March 28, 2011 ZOOMED In: Sub 3um Columns Produce Sharp, Efficient Peaks for LC/MS/MS – Toxicology Sample

Poroshell 120 EC-C18, 2.1 x 100 mm, 2.7 µm 1.8-µm particle column: tallest, sharpest peaks, 2.7-µm particle column has ZORBAX RRHT Eclipse Plus C18, 2.1 x 100 mm, 1.8 µm similar results 3.5-µm column produces slightly shorter peaks, ZORBAX Eclipse Plus C18, 2.1 x 100 mm, 3.5 µm 5-µm column generates significantly broader, shorter ZORBAX Eclipse Plus C18, 2.1 x 100 mm, 5 µm peaks List of Compounds in Toxicology Mixture

Amphetamine Phentermine Trazodone Methamphetamine Proadifen Cocaine Meperidine Oxazepam MDEA MDMA Lorazepam MEA Diazepam Temazepam O,O'-Diacetylmorphine Nitrazepam Clonazepam Alprazolam Verapamil Strychnine

Confidentiality Label 24 March 28, 2011 Peak Width vs. Column Particle Size in LC/MS Toxicology Sample

5um particles provide very broad peaks. Peak Width decreases with particle size.

Confidentiality Label 25 March 28, 2011 Comparison of Separation of Toxicology Sample by MS/MS on Columns of Various Particle Sizes

• Agilent ZORBAX Eclipse Plus C18, 2.1 x 100mm, 5um Comparison • Agilent ZORBAX Eclipse Plus C18, 2.1 x 100mm, 3.5um columns: • Agilent ZORBAX Eclipse Plus C18, 2.1 x 100mm, 1.8um • Agilent Poroshell 120 EC-C18, 2.1 x 100mm, 2.7um

• from sharpest peaks to broadest peaks Results: • 1.8um > 2.7um > 3.5um > 5um

• There is a sensitivity benefit for LC/MS with small Conclusion: particle columns

Confidentiality Label 26 March 28, 2011 Different Scan Rates on MS

•Green tea sample on UV, MS and MS/MS •MS scan rates in scan mode •MS dwell time in SIM mode •MS/MS cycle times, dynamic MRM Comparison of different UV data collection rates and different MS scan rates in Scan mode Column: ZORBAX RRHD SB-C18, 2.1 x 100mm, 1.8um, 1200 bar Sample: Green Tea UV Data Collection Rate MS Scan Rate 0.12 s x106 mAU 25 ms 300 1 1 200 PW1/2=0.018 5 PW1/2=0.024 100 0 S/N=39 0 0.25 0.5 0.75 1 1.25 1.5 1.75 min x106 mAU0.25 s 50 ms 300 1 1 200 PW1/2=0.018 5 PW1/2=0.024 100 0 S/N=43 0 0.25 0.5 0.75 1 1.25 1.5 1.75 min x106 mAU0.5 s 100 ms 300 1 1 200 PW1/2=0.019 5 PW1/2=0.027 100 S/N=86 0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 min x106 mAU1.0 s 250 ms 1 1 300 5 200 PW1/2=0.025 PW1/2=0.035 100 S/N=118 0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 min x106 500 ms mAU2.0 s 1 1 300 PW =0.042 5 PW =0.049 200 1/2 1/2 100 S/N=109 0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 min 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 Comparison of different UV data collection rates and different MS dwell times in SIM mode Column: ZORBAX RRHD SB-C18, 2.1 x 100mm, 1.8um, 1200 bar Sample: Green Tea UV Data Collection Rate MS SIM Dwell Time 0.12 s x106 mAU 1 ms 300 1 1 1 200 PW1/2=0.018 PW1/2=0.024 100 0 S/N=113 0 0.25 0.5 0.75 1 1.25 1.5 1.75 min x106 mAU0.25 s 10 ms 300 1 1 1 200 PW1/2=0.018 PW1/2=0.027 100 0 S/N=198 0 0.25 0.5 0.75 1 1.25 1.5 1.75 min x106 mAU0.5 s 25 ms 300 1 1 1 200 PW1/2=0.019 PW1/2=0.031 100 S/N=714 0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 min x106 mAU1.0 s 50 ms 1 1 300 1 200 PW1/2=0.025 PW1/2=0.039 100 S/N=744 0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 min 6 2.0 s x10 75 ms mAU 1 1 300 PW =0.042 1 PW =0.050 200 1/2 1/2 100 S/N=277 0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 min 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 Comparison of different UV data collection rates and different MS/MS cycle times in dynamic MRM mode Column: ZORBAX RRHD SB-C18, 2.1 x 100mm, 1.8um, 1200 bar Sample: Green Tea UV Data Collection Rate MS/MS dMRM Cycle Time 0.12 s x105 mAU 60 ms  Min/Max Dwell Time: 11.50/26.50 ms 300 1 1 1 200 PW1/2=0.018 PW1/2=0.022 100 0.5 S/N=2507 0 0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 min x105 mAU0.25 s 80 ms  Min/Max Dwell Time: 16.50/36.50 ms 300 1 1 1 200 PW =0.018 PW =0.022 1/2 0.5 1/2 100 S/N=2578 0 0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 min x105 mAU0.5 s 100 ms  Min/Max Dwell Time: 21.50/46.50 ms 300 1 1 1 200 PW =0.019 PW1/2=0.023 1/2 0.5 100 S/N=3188 0 0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 min x105 mAU1.0 s 200 ms  Min/Max Dwell Time: 46.50/96.50 ms 1 1 300 1 200 PW =0.028 PW1/2=0.025 0.5 1/2 100 S/N=5702 0 0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 min x105 500 ms  Min/Max Dwell Time: 121.50/246.50 ms mAU2.0 s 1 1 1 300 PW =0.042 PW1/2=0.050 200 1/2 0.5 100 S/N=2389 0 0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 min 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 Observations on “Data Rate” Increases in MS Scan Mode, SIM Mode, and Dynamic MRM Mode Sharper and narrower peaks in UV and MS – up to a point where additional data points are not necessary MS shows sharper peaks, but declining S/N due to increased noise

Confidentiality Label 31 March 28, 2011 50X Signal:Noise Improvement with MS/MS detection over an MS scan – Green Tea Analysis Column: ZORBAX RRHD SB-C18, 2.1 x 100mm, 1.8um, 1200 bar x106 MS Scan Mode, 250 ms Scan Time 1 1 A: 0.2% acetic acid in water 6 B: acetonitrile

4 1 mL/min Time (min) 0.0 0.5 2.0 2 S/N=118 %B 10 15 21 Stop time: 2.0min Post run time: 2.0 min x105 MS SIM Mode, 50 ms Dwell Time 1 uL injection of green tea 1 1 standard: 6 ng/mL each in water 8 TCC: 40 oC 6 Agilent ZORBAX RRHD SB-C18, 4 2.1 x 100 mm, 1.8 um 2 S/N=744 MS, Pos SIM: 171, 307, 195, 291, 459, 443 MS/MS, dMRM transitions: x104 MS/MS1 dynamic MRM Mode, 200 ms Cycle Time 1 GA: 171109, 127 6 (E)GC: 307139, 121 5 (E)C: 291139,123 4 3 Caff: 195 138, 110 2 (E)GCG: 459139, 289 1 S/N=5702 (E)CG: 443139, 123

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 Counts vs. Acquisition Time (min) Comparison of Mobile Phases for LC/MS Separations S/N comparison of different acidic mobile phase additives • Acetic acid • Formic acid • Triflouroacetic acid Comparison of acid and volatile salt/acid

Confidentiality Label 33 March 28, 2011 Comparison of S/N with acetic acid, formic acid and TFA in LC/MS Green Tea Standards Sample: 3 µL of 3.3 µg/mL each of gallic acid, gallocatechin, epigallocatechin, catechin, caffeine, epicatechin, epigallocatechin gallate, gallocatechin gallate, epicatechin gallate, catechin gallate in

H2O/CH3CN Column: Poroshell 120 SB-C18, 2.1 x 100mm, 2.7um x10 2 1 0.2%1 HCOOH 1 0.9 • 0.2% acetic acid

0.8

0.7 yields the least 0.6 0.5 S/N=111 amount of ion 0.4 0.3 suppression and 0.2 0.1 strongest signal 0 x10 2 1 for all 0.2%1 CH COOH 1 0.9 3 0.8 • TFA results in the 0.7 0.6 most significant 0.5 S/N=155 0.4 ion suppression 0.3 0.2 0.1 • Peak shape and 0 x10 2 selectivity remain 1 1 1 0.02% CF3COOH 0.9 the same for all 0.8 0.7 mobile phases 0.6 0.5 S/N=68 0.4

0.3 0.2 0.1

0 Comparison of Ammonium Acetate and Acetic Acid Mobile Phases in Green Tea Analysis Sample: 3 µL of 3.3 µg/mL each of gallic acid, gallocatechin, epigallocatechin, catechin, caffeine, epicatechin, epigallocatechin gallate, gallocatechin gallate, epicatechin gallate, catechin gallate in H O/CH CN 2 3 Column: Poroshell 120 SB-C18, 2.1 x 100mm, 2.7um

2 x10 0.2% CH3COOH 1 • 0.2% acetic acid 1 1 produces ≈5x more 0.75

intense signal 0.5 • 10 mM acetate buffer 0.25 S/N=155 prepared from the

ammonium salt, shows 0 significant ion 2 x10 10 mM CH3COONH4, pH 3.6 (adjusted to pH with 10 mM CH3COOH) suppression 1 1 1 • Peak shape and 0.75 selectivity appear similar with both mobile 0.5 phases 0.25 S/N=33

0 Are the Small Particle Columns Reliable?

Yes! Challenged with dirty samples • Lifetime • Reproducibility

Confidentiality Label 36 March 28, 2011 Long Lifetime with Poroshell 120 2.7um Column >1800 injections at 550 bar no performance change

Life Time Test with Unfiltered, Undiluted Freshly Brewed Green Tea Sample 0.05

0.04 Caffeine 0.03

Height,s System Pressure, Epicatechin

2 / 1 0.02 Epicatechin 0.01 Gallate 0 0 200 400 600 800 1000 1200 1400 1600

Peak WidthPeak at Number of Injections

A: 0.2% HCOOH in H2O

B: 0.2% HCOOH in CH3CN

0.833 mL/min EGCG mAU Time 0.00 1.25 2.50 800 EGC Pmax=550bar %B 10 15 27 o 600 40 C

Caf. Column: Agilent Poroshell 120 SB-C18, 2.1 x 100 mm, 2.7 µm

400 Sig=210,4nm, Ref=Off ECG

GC 2-µL, 3-mm micro flow cell (PN G1315-60024)

200 EC GA

GCG Sample: 2 µL of freshly brewed green tea (brewed from a commercial CG C tea bag in 6 oz of initially boiling water for six minutes) 0

0.5 1 1.5 2 2.5 3 3.5 min Reproducibility for 110 Injections (10 fmol SIS Peptides and 2.5 µg Plasma Digest On-column) Response Ret. Time Protein %RSD %RSD Adiponectin: 9.8 0.13 IFYNQQNHYDGSTGK Antithrombin-III : Plasminogen LFLEPTR 4.7 0.16 DDLYVSDAFHK Apolipoprotein A-II precursor: 6.7 0.12 2.2% RSD SPELQAEAK n=110 Apolipoprotein C-III: 2.3 0.08 GWVTDGFSSLK Ceruloplasmin : 9.6 0.14 EYTDASFTNR Heparin cofactor II: 6.1 0.15 TLEAQLTPR Histidine-rich glycoprotein: 3.4 0.02 DGYLFQLLR Kininogen-1: 3.3 0.13 4.7% RSD, n=4 TVGSDTFYSFK L-selectin: 9.5 0.15 AEIEYLEK Plasminogen: 2.2 0.13 7.9% RSD, n=4 LFLEPTR Vitamin D-binding protein: 12.3% RSD, n=4 3.0 0.12 THLPEVFLSK von Willebrand Factor: 9.5 0.15 ILAGPAGDSNVVK The samples were provided by Derek Smith and Christoph H. Borchers from the UVic-Genome BC Proteomics Centre Conclusions

Small particle size columns – including sub 2um and superficially porous particle columns – enhance MS and MS/MS results. Improvements in sensitivity, resolution and peak capacity with small particle columns improve LC/MS results. High resolution, small particle columns are very useful if the sample contains isomers or compounds with the same molecular weight. The mobile phase can be selected to provide optimum MS results – peak shape

Confidentiality Label 39 March 28, 2011