Important Qualities of Mobile Phase Additives for LC-MS Applications

What are the important qualities of mobile phase additives for LCMS applications? Dannie Mak and Stephen Roemer Thermo Fisher Scientific – Global Chemicals, Fair Lawn, NJ LC_TS_EU_823_14 Chemicals The mobile phase composition plays a critical role in the success of an LC-MS experiment. Indeed, solvents designated “LC-MS grade” should provide low mass background, minimal organic contamination, and minimal metal content to fulfi ll the high purity need of LC-MS. It is common practice in LC-MS to add certain volatile chemicals to the mobile phase solvents in order to provide stable analyte ionization and better chromatography. Different additives are available and which one to use depends on the nature of the analyte and on the LC and MS conditions. Regardless of the choice of additive, it must be highly pure to avoid introduction of extraneous mass signals and it should also be application tested. The Optima™ LC/MS product line includes essential neat additives such as A113 Acetic Acid (AA), A116 Trifluoroacetic Acid (TFA), and A117 Formic Acid (FA), coupled with their ready-to-use aqueous and organic blends. These mobile phase additives are use-tested, and our data will show that their mass baselines are very low for positive mode in TIC with single quadrupole and ion trap mass detector, very low LC-UV response using diode array detector, and exceptionally low metal ion content with ICP-MS which makes MS interpretation easier due to reduced metal adduct formation. Additionally, for proteomics research these additives and blends are proteasefree which ensures that target peptides or proteins are not destroyed by these pervasive hydrolyzing enzymes. Single Quadrupole MS: LC Conditions: • LC System: Agilent 1100 Series LC • Column: ZORBAX 300 SB-C8, 2.1 mm x 150 mm x 3.5 micron • Flow Rate: 0.425 ml/min • Mobil Phase: 0.1% TFA or FA in Acetonitrile or Water • Gradient: Various • Detector: Diode Array Detector • Two cycles of gradient were run. Only data from the second circle was used MS Conditions: • MS System: SL Model Single Quadrupole Mass Spec Detector • Ionization Mode: ESI, positive and negative • Scan Range: 15 – 1500 m/z • Vf: 70 V • Capillary Voltage: 3000V LTQ-XL Ion Trap MS: LC Conditions: • LC System: Thermo Scientifi c Accela U-HPLC • Column: Hypersil® GOLD, 2.1 mm x 50 mm x 1.9 micron • Flow Rate: 400 μl/min • Mobil Phase: 0.1% TFA or FA in Acetonitrile or Water • Gradient: 80% A to 20% B (organic) • Detector: Diode Array Detector MS Conditions: • MS System: LTQ XL Ion Trap Mass Spec Detector • Ionization Mode: ESI, positive and Pos-SIM • Scan Range: 50 – 2000 m/z Mobile Phase Solvents and Additives: • Fisher Chemical Optima® LC/MS Acetonitrile (A955) and Water (W6) • Optima LC/MS Acetic Acid (A113), Formic Acid (A117) and Trifl uoroacetic Acid (A116) At 280 nm, the Fisher Chemical FA-water blend has fewer impurity peaks and lower peak heights compared to the competitor’s material. QUESTION: Frequently, researchers are analyzing small amounts of target compound which requires a mobile phase with fl at baseline and low background noise. What is the general baseline at 210 nm for Fisher Chemical LCMS grade 0.1% formic acid in acetonitrile? At 210 nm, the Fisher Chemical FA-acetonitrile blend has a fl atter and lower baseline compared to Vendor J. QUESTION: Formic acid is frequently added to the LCMS mobile phase because of its ability to provide protonation ions for analyte in mass detector. This enhances chromatography performance in terms of better peak shape, better separation of the analytes, and stable signal detection. With respect to mass baseline, how does the Fisher Chemical FA-modifi ed mobile phase compare to another leading brand? MASS BACKGROUND IN TIC POSITIVE MODE USING 0.1% FA IN WATER (SINGLE QUADRUPOLE MS) Three lots of Fisher Chemical FA-modifi ed aqueous mobile phase produced consistently the fl attest and lowest background noise in TIC compared to Vendor J. MASS BACKGROUND IN TIC POSITIVE MODE USING 0.1% FA IN ACETONITRILE (SINGLE QUADRUPOLE MS) The chromatography shown here illustrates a lower baseline for the Fisher Chemical FAmodified organic mobile phase compared to the competitor’s material. QUESTION: What are the chromatographic benefi ts of modifying the LCMS mobile phase with formic acid? EFFECT OF ANALYZING THE TARGET COMPOUND WITH AND WITHOUT 0.1% FORMIC ACID IN THE MOBILE PHASE BY SINGLE QUADRUPOLE MS (FULL SCAN) The target compound (reserpine, MW = 609.68 ) elutes faster and has better peak shape (narrower) when FA is added to the mobile phase (bottom two chromatograms). Column a) and b) are duplicates of reserpine elution with no additives in the mobile phase and columns c) and d) are reserpine elution with 0.1% formic acid added to the mobile phase. The type of signal collected is listed by row (Row 1 = MS full scan, Row 2 = MS extracted ion, Row 3 = MS SIM mode, and Row 4 = LC-254 nm). When the mobile phase is not modifi ed by formic acid, the eluted reserpine peak is wider and spikier in shape, especially for the MS extracted mass ion (Row 2). In contrast, when formic acid is added to the mobile phase the reserpine peak is sharper, narrower in width, and has a more symmetrical peak shape. EFFECT OF ANALYZING THE TARGET COMPOUND WITH AND WITHOUT 0.1% FORMIC ACID IN THE MOBILE PHASE BY LTQ-XL ION TRAP MS No FA No FA Add FA Add FA a) b) c) d) Row 1 MS Full Row 2 MS Extract Ion Row 3 MS SIM Row 4 LC-254 nm QUESTION: Frequently, researchers are analyzing small amounts of target compound which requires a mobile phase with fl at baseline and low background noise. What is the general baseline at 210 nm for Fisher Chemical LCMS grade 0.1% formic acid in acetonitrile? METAL IMPURITIES IN FORMIC ACID (FA) Although some vendors may claim a certain specifi cation for metal content, very often an actual measurement of the product reveals a different value. For example, the actual sodium content in Vendor J’s product is about 75 ppb, rather than claimed 50 ppb maximum. In contrast, Fisher Chemical’s 0.1% FA in water has the lowest metal content of the tested materials. QUESTION: QUESTION: For proteomics applications, are the Fisher Chemical LCMS mobile phase blends protease free? PROTEASE TEST FOR 0.1% FORMIC ACID IN WATER Fisher Chemical Optima LC/ MS aqueous blends are FREE of PROTEASE. This specifi cation is important for proteomics research since peptides/proteins could be degraded if the mobile phase solution is contaminated with protease. 1) BSA, Negative Control 2) BSA + Protease, Positive Control 3) BSA + 0.1% FA sample 4) BSA + 1x SPK Protease + 0.1% FA sample 5) BSA + 2x SPK Protease + 0.1% FA sample 6) BSA + 5x SPK Protease + 0.1% FA sample Result: No protease is detected in 0.1% FA aqueous blend Note: 1μl of 0.1% FA aqueous blend added to reactions 3 – 6. QUESTION: Acetic acid (AA) is a small, volatile organic acid that is commonly added to the LCMS mobile phase similar to formic acid, but with a different pH environment as the analyte may require. In contrast, trifl uoroacetic acid (TFA) is known to suppress MS ionization although use of a low concentration of TFA can enhance retention and improve peak shape of a product during analytical separation. Compared to FA, how does AA and TFA and their associated mobile phase blends perform with respect to mass baselines, metal content, and protease impurity? At LC-280 nm, the Fisher Chemical 0.1% TFA in water has fewer impurity peaks and lower baseline compared to Vendors’ material. At LC-280 nm, the Fisher Chemical acetic acid has fewer impurity peaks than Vendor S material. PROTEASE TEST FOR 0.1% TFA IN WATER MASS BACKGROUND IN ES+ MODE USING 0.1% ACETIC ACID IN WATER (SINGLE QUADRUPOLE MS) Fisher Chemical 0.1% acetic acid in water has a lower average mass intensity compared to Vendor S material. METAL IMPURITIES IN TRIFLUOROACETIC ACID (TFA) The chromatography shown here illustrates a lower average mass intensity for Fisher Chemical 0.1% acetic acid in water compared to Vendor S. The actual sodium content in Vendor S TFA is about 665 ppb, rather than claimed 50 ppb maximum. In contrast, Fisher Chemical TFA has the lowest metal content of the tested materials. This specifi cation is important for proteomics research since peptides/proteins could be degraded if the mobile phase solution is contaminated with protease. 1) BSA, Negative Control 2) BSA + Protease, Positive Control 3) BSA + 0.1% TFA sample 4) BSA + 1x SPK Protease + 0.1% TFA sample 5) BSA + 2x SPK Protease + 0.1% TFA sample 6) BSA + 5x SPK Protease + 0.1% TFA sample Result: No protease is detected in 0.1% TFA aqueous blend. Note: 1μl of 0.1% TFA aqueous blend added to reactions 3 – 6. Abstract Methods and Instrumentation Results Key features of Fisher Chemical Optima LC/MS Acetic Acid (A113), Trifl uoroacetic Acid (A116), Formic Acid (A117), and their associated mobile phase blends: • Low mass spectrometry background noise • Lowest impurity background using diode array detection (LC-UV)