Evaluation of UHPLC System Performance Under High Throughput Conditions. William Hedgepeth, Masatoshi Takahashi, Shimadzu Scientific Instruments
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1 Evaluation of UHPLC System Performance Under High Throughput Conditions William Hedgepeth, Masatoshi Takahashi, Shimadzu Scientific Instruments
2 Introduction The use of UHPLC technology has continued to increase in LC applications as a way to reduce sample analysis turnaround times. Maintaining or increasing resolution of faster separations has been made possible with the use of small particle column technology. UHPLC often provides run times of less than one minute with peak widths of less than 200 milliseconds, which have created challenges for system performance and detection methods. A variety of applications in pharmaceutical and environmental areas with small particle columns will be presented that will demonstrate the throughput and performance that can be obtained with current UHPLC technology.
3 UHPLC System Evaluated Nexera LC-30A system Max pressure: 130 MPa (18,850 psi) up to 3 ml/min Injection technique: direct and loop injection methods Detector speeds: UV, Fluorescence 10 msec (100 Hz) MS 15,000 u/sec
4 Peak Capacity Evaluation Peak capacity measures the resolving power of a UHPLC system and is based on average peak width and gradient time. A high peak capacity of over 30 peaks per minute could be obtained. mau 17.5 Column : Zorbax Eclipse RRHD C18 2.1mm, 100mm, 1.8um 15.0 Trypsin digested BSA Mobile phase : A : 0.03 % TFA in water B : 0.03 % TFA in acetonitrile 12.5 Gradient : B 5% 40 % (8 min) 10.0 Mixer Flow rate : 180 ul : 0.9 ml/min 7.5 Column temp. Detection : 40C : 214 nm 5.0 Sample : Trypsin digested BSA 2.5 (total of 1 pmol / ul) min Peak capacity: 244 in 8 minutes
5 Impurity A Impurity B Diazepam Diazepam Impurity Evaluation Wide dynamic range and high sensitivity Main compound: diazepam Two compounds (impurities) were spiked into the diazepam solution. The impurity samples were prepared to be 0.5, 0.05, % w/w relative to the main compound. Column Mobile phase Gradient Flow rate Column temp. Detection : Zorbax Eclipse RRHD C18 2.1mm, 100mm, 1.8um : A : water B : acetonitrile : B 25% 40 % (0.1 min) 70 % (3.0 min) : 0.8 ml/min : 30C : 240 nm mau min 6 mau ZOOM 0.05 %w/w min
6 Impurity A Impurity B Diazepam Sensitivity Evaluation UHPLC systems can provide higher sensitivities since sharper peaks are obtained. Peak area RSDs were evaluated at low-level impurity concentrations down to 0.005%. The wide dynamic range in UHPLC systems allows simultaneous assay and impurity analysis. Repeatability of peak area, (% RSD, n=6, 6 ul injection) uv LOD : % (9 pg on column) impurity content (%) Impurity A Impurity B % w/w 0.05 % w/w % w/w min
7 Ultra-high Pressure Evaluation Theoretical plate number is not compromised at ultra-high pressures. 175 mau min 125 mau um 150mm L 53 Mpa (7,685 psi) TPN at peak 7 = Rs (1.2) = 5.01 Width (1) 50% = min 1.8 um 300mm L 115 Mpa (16,675 psi) TPN at peak 7 = Rs (1.2) = 7.14 Width (1) 50% = min min Zorbax eclipse plus C18 ODS (2.1mmi.d. 150mm, 1.8 um) 0.5 ml/min Water/acn = 20/80 50C 245nm 1. Acetophenone 2. Propiophenone 3. Butyrophenone 4. Valerophenone 5. Hexanophenone 6. Heptanophenone 7. Octanophenone Zorbax eclipse plus C18 ODS (2.1mmi.d. 300mm, 1.8 um) 0.5 ml/min Water/acn = 20/80 50C 245nm
8 Cortisone Cortisol Cortisone Cortisol Use of Methanol Mobile Phase Higher pressure capability allows alternate solvent usage for selectivity purposes Analysis of cortisone and cortisol in Urine (each 5 ppm, spiked) mau min mau Methanol mobile phase Acn : 69 MPa Methanol : 108 MPa min Column Mobile phase Gradient Flow rate Pressure Column temp. : 40 C Detection Column Mobile phase Gradient Flow rate Pressure : Zorbax Eclipse RRHD C18 2.1mm, 100mm, 1.8um : A :0.1 % formic acid in water B : acetonitrile : B 10% 60 % (3.5 min) : 0.6 ml/min : 69 MPa (10,005 psi) : 245 nm Preparation of urine sample (cortisone / cortisol : 5ppm) Acetonitrile addition Centrifugation (12,500 rpm, 3min ) Evaporation and reconstitution by water Column temp. : 40 C Detection : Zorbax Eclipse RRHD C18 2.1mm, 100mm, 1.8um : A : 0.1 % formic acid in water B : methanol : B 30% 90 % (3.0 min) : 0.6 ml/min : 108 MPa (15,660 psi) : 245 nm
9 Analgesics Ultra-Fast Analysis Analgesics area RSD (100 ug/ml Inj: 1 ul) uv Acetaminophen 2 Caffeine 3 2-Acetamidophenol 4 Acetanilide 5 Acetylsalicylic acid 6 Phenacetin 7 Salicylic acid min Column : Zorbax Eclipse RRHD C18 2.1mm, 100mm, 1.8um Mobile phase : A: water (0.1 % H3PO4) /B :acetonitrile Gradient : B 5% 45 % (0.20 min) 60 % (0.80 min) Flow rate : 1.1mL/min Column temp. : 40 C Detection : 240 nm Pressure : 85 MPa (12,325psi) Compound area %RSD Acetaminophen 0.15 Caffeine Acetamidophenol 0.21 Acetanilide 0.15 Acetylsalicylic acid 0.17 Phenacetin 0.17 Salicylic acid 0.19
10 Low-volume Injection Evaluation Zorbax Eclipse Plus RRHD 1.8um 50mmL. X 2.0mmI.D. MP : (A) 50mmol/L Ammonium Acetate Buffer(pH 4.7) /Ethanol=100/5 (B) ACN Prog. : B.Conc. 35%(0min)->45%(1-1.25min) Flow Rate : 1.1mL/min (74Mpa) Inj : 0.5uL Temp : 40deg Det. : 235nm Resp. : 10msec (100 Hz) RT (%RSD) area%rsd Peak min (0.274%) 0.462% Peak min (0.187%) 0.330% Peak min (0.146%) 0.398% Peak min (0.136%) 0.521% Peak min (0.106%) 0.445% Peak min (0.101%) 0.390% Peak min (0.086%) 0.310% Peak min (0.059%) 0.422% Peak min (0.048%) 0.478% Peak min (0.038%) 0.252% Peak min (0.041%) 0.332% Peak min (0.071%) 0.382% Peak min (0.077%) 0.455%
11 UHPLC with Fluorescence 1ºC temperature fluctuation can result in a 5% change in peak intensity Temperature-controlled flow cell with cooling allows improved RSD performance With Cell Temperature Control No Cell Temperature Control : Room temperature 25 ºC : Room temperature 30 ºC : Room temperature 25 ºC : Room temperature 30 ºC Area RSD 0.3% Change of Area 0.64% Area RSD 6.3% Change of Area % min min
12 Quinolones (Fluorescence Data) Zorbax Eclipse Plus 1.8um 50mmL. X 3.0mmI.D. MP : (A) 0.05% Formic acid aq. (B) 0.1% Formic acid / ACN Prog. : B.Conc. 8%->16%(0.75min) ->36%(1-1.5min) ->95%(2min) Flow Rate : 2.0mL/min Inj : 1uL Temp : 55deg Det. : Ex.295nm Em.455nm 10msec GainX4 (0-1.05min) Ex.325nm Em.365nm, 100msec GainX16 (1.05-3min) ppmX1μL (n=6) RT %RSD Area %RSD Peak % 0.231% Peak % 0.385% Peak % 0.118% Peak % 0.138% Peak % 0.095% Peak % 0.117% Peak % 0.214% Peak % 0.278% Peak % 0.057% Peak % 0.210% Peak % 0.153% 1.Norfloxacin 2.Ofloxacin 3.Ciproploxacin 4.Danofloxacin 5.Enrofloxacin 6.Orbifloxacin 7.Sarafloxacin 8.Difloxacin 9.Oxolinic acid 10.Nalidixic acid 11.Flumequine
13 UHPLC-MS Mass spectrometer requirements for UHPLC: What makes an MS instrument suitable for UHPLC? The ability to acquire data at high speed without sacrificing data quality. The three things that enable high-speed analysis: The ability to perform scan measurement at high speeds scanning up to 15,000 u/sec The ability to switch between positive and negative ion measurement at high speed polarity switching in 15 msec High sensitivity in high-speed measurement design to prevent loss of sensitivity in high-speed analysis
14 1 Carbofuran 4 Daimuron 5 Iprodione 6 Carpropamid 2 Bentazone 3 Tryclopyr 4 Daimuron 6 Carpropamid UHPLC/MS Reproducibility in SIM mode Polarity switching Polarity switching Positive Ion SIM Negative Ion SIM Positive Ion SIM (x1,000,000) (x1,000,000) (1.00) (1.00) (1.20) (6.00) (15.00) (3.50) Positive Ion SIM (1.00) (2.00) Negative Ion SIM min min SIM 8 CH 0.02 s each CH, Flow Rate: 1.2 ml/min Column: XR-ODS 2.2 um n=6 Positive Ion %RSD R.T %RSD Area n=6 Negative Ion %RSD R.T %RSD Area
15 UHPLC with High-speed MS Nexera-LCMS-2020 Analysis of 30 pharmaceuticals by high-speed polarity switching (15 msec) and high-speed scanning technology (15,000 u/sec) Column : Zorbax Eclipse RRHD C18 2.1mm, 50mm, 1.8um Mobile phase : A : 0.1 % formic acid in water / B : 0.1 % formic acid in acetonitrile Gradient : B 3% 95 % (0.50 min) Flow rate : 1.8mL/min Column temp. : 50C Detection : ESI (+/-), LCMS-2020, Scan speed : u/sec Positive 1. Atenolol (267) 2. Procaine (237) 3. Lidocaine (237) 4. Atropine (290) 5. Yohimbine (355) 6. Chlorpheniramine (275) 7. Propranolol (260) 8. Alprenolol (250) 9. Tetracaine (265) 10. Diphenhydramine (256) 11. Doxepin (280) 12. Dipyridamol (505) 13. Desipramine (267) 14. Imipramine (281) 15. Nortriptyline (264) 16. Amitriptyline (278) 17. Dibucaine (344) 18. Verapamil (455) 19. Reserpine (609) 20. Carbamazepine (237) 21. Isopropylantipyrine (231) 22. Alprazolam (309) 23. Trizolam (343) 24. Cilostazol (370) 25. Nifedipine (347) 26. Diazepam (285) 27. Warfarin (309) Negative 1. Cefuroxime (423) 2. Chloramphenicol (321) 3. Nitrendipine (359)
16 High-speed Scan Analysis by LCMS-2020 Nexera-LCMS-2020 High-speed scan (15000 u/sec) doesn t miss any UHPLC peaks Lidocaine 2. Desipramine 3. Dibucaine 4. Reserpine 5. Butorphanol 6. Dextromethorphan 7. Pentazocine 8. Dextrophan 9. Chlorphenyramine 10. Diethylpropion 11. Ketamine 12. Phenmetrazine 13. Verapamil 14. Diphenhydramine 15. Buprenorphine Column Mobile phase Gradient Flow rate Column temp. Detection Scan speed : Zorbax Eclipse RRHD C18 2.1mm, 50mm, 1.8um : A :0.1 % formic acid and 10mM ammonium acetate in water B : acetonitrile : B 10% 75 % (0.36 min) : 1.7mL/min (Pressure : 100 MPa) : 50C : ESI (+), LCMS-2020 : u/sec
17 Conclusions Reproducibility data with UHPLC conditions can match or exceed those of conventional HPLC conditions. Acquisition rates of 10msec (100 points per second) for UV and fluorescence detectors allow accurate quantitation of UHPLC peaks. MS scanning speeds of 15,000 u/sec and polarity switching speeds of 15 msec match the needs of narrow UHPLC peaks.
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