Potential sources of contamination on Mass Spectrometers and suggested cleaning procedures

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1 Potential sources of contamination on Mass Spectrometers and suggested cleaning procedures Purpose: This document provides information on potential sources of background contamination commonly encountered on mass spectrometers, which can degrade the quality of results by increasing the background, reducing sensitivity. 1. Possible background ions Name: Polyethylene glycol (PEG). Mass spectrum: Usually seen in ESI + as a series of ions separated by 44Da. Sources: 1) Organic solvents e.g. methanol, water, acetonitrile, isopropanol. 2) Dermatological creams 3) Plastic gloves, protein extraction detergents (e.g. Triton X100, X114), 4) Glassware detergents 5) Cutting solutions 6) MS calibration solutions 7) Column manufacture _50ACN_L02_B1 120 (2.284) Cm (120:140) : TOF MS ES+ 176 PEG-LIKE CONTAMINATION 50:50 ACN:H2O +0.1% formic acid Combined 21 spectra % m/z Figure 1: Typical PEG contamination 34301_1 1 of 9

2 Name: Phthalates Mass Spectrum: Seen as [M+H] + in ESI + at m/z = 391, 419 and 447. Generally attributed to Di-isooctylphthalate, Dinonylphthalate, Diisodecyl phthalate respectively. Can form adduct ions with sodium, potassium and ammonium ions. e.g. for Di-isooctylphthalate [M+Na] + = 413, [M+K] + = 429, [2M+NH 4 ] + = 798, [2M+Na] + = 803, depending on conditions. Sources: 1) Can be extracted from plastics e.g.. Tubing, plastic bottles, plastic vials, vial caps. 2) Can be airborne and enter solvents by absorption. Notes: A wide range of phthalate compounds are used as plasticizers in many common plastics. Ion signals arising from these species can be more intense when utilising high aqueous conditions due to increased solubility. Figure 2: Typical pthalate background ions Name: Metal ions (e.g.. Na +,K +,Fe 2/3+,Cu 2+, Pt + ) Mass Spectrum: Cause adduct formation e.g.. [M+Na] +, [M+K + ], etc. Fe 3+ can form adducts with varying numbers of acetates from acetic acid or acetate buffers to give characteristic ESI+ ions at m/z = and Sources: 1) Solvents 2) Buffers 3) Additives (e.g. acetic/formic acid) 4) Metal components 34301_1 2 of 9

3 Notes: Solvent manufacturers usually state trace metal content of their solvents on the side of the bottle, or on the MSDS sheet. These can vary a great deal between manufacturer and also the particular grade of solvent. E.g. HPLC grade usually has a lower metal content than other grades. Name: Siloxanes e.g.. Polydimethylcyclicsiloxanes of monomeric unit (Si(CH 3 ) 2 O) n Mass Spectrum: Observed at m/z = 445, 519, 593, 667, 741. Generally can be attributed to decamethylsiloxane, dodecamethylsiloxane, tetradecamethylsiloxane, hexadecamethylsiloxane, octacdecamethylsiloxane respectively. Additional masses can be observed at m/z = 429 and 503. Also seen are ammonium adducts (+17Da) and ions as a results of a methyl group loss. Sources: 1) Silica capillary, HPLC column packing materials etc 2) Ubiquitous airborne contaminants Notes: Generally seen more often under NanoFlow conditions. Procedures for removal of these background ions are limited due to the overwhelming presence in the atmosphere. Detuning the probe positions can reduce background ions while not severely reducing sample signal. A higher concentration of nitrogen in the source region can also reduce siloxane production at the probe tip. The paper: Schlosser A, Volkmer-Engert R, J. Mass Spectrom, 2003, 38, p is a good reference for these ions. Figure 3: Typical siloxane background ions on a Nanospray source _1 3 of 9

4 2. Minimizing background ions a) Solvents and additives Use the highest purity solvents HPLC grade or better i. Solvents 1. The current solvents in use at Waters MS Technologies Atlas Park are Water (Fisher W/0106/17) Methanol (Fisher M/4058/17) and Acetonitrile (Sigma 34888). 2. Some HPLC grade solvents contain PEG. PEG is not seen by a UV detector, but will be observed using an MS. Always read the label. 3.Some HPLC grade solvents contain metal ions. 4. Fresh solvents are better than previously opened bottles. 5. Additives should also be pure. 90% formic acid solution contains 10% other compounds! Analytical grade (>99%) is recommended. ii. Water can be a major source of contamination 1. On-line water systems (Milli-Q) must be maintained. 18 MOhm water is not free of organic contamination. Organic filters and UV lights increase the purity for HPLC use. 2. Bottled water may not be any better than water from Milli-Q. After a bottle has been opened, it will accumulate contaminates by absorption from the air, through any secondary container, or via general use (e.g.. pipettes etc). Storage of water in plastic containers is NOT recommended. Water can be further purified by passing it through a clean C18 column to trap organic materials. This can be done on-line in a high-pressure binary gradient system with a C18 guard column. Additives like acetic acid can contain a significant amount of iron. E.g. 100 ppb (parts per billion) may sound like a low level. However, 100 ppb = 100 ng/l or 100 pg/µl. Most current MS systems have specifications at much lower concentrations than this. E.g.. Reserpine 1pg/µL for many signal:noise specifications. Presence of metal ions in acetic acid can form adducts which produce intense peaks in ESI+ spectra. The use of additives can be a balance between the increase in sensitivity of the analyte compounds against the increase in the background ion intensities. b) Sample and solvent containers The following items can be sources of background ions i. Vials, caps, well plates 1. Waters vials are quality controlled. Brand X may not be as clean 2. Vial caps with septa may contain plastics or adhesives 3. Well plate glue-on foil covers may leach adhesives ii Glassware 1. Washing in a common dishwashing facility can contaminate glassware with detergent residues (PEG-like compounds) and surfactants. 2. Rinse only with mobile phase quality solvents that will be used _1 4 of 9

5 iii. Plastic containers or tubing Storage of solvents or water in plastic is not recommended as compounds may leach out of the container into the contents (e.g. dioctylphthalate contamination). c) Sample and sample matrix Sample matrix may contain salts and other unwanted materials. The solubility of the matrix must be considered when preparing needle wash solutions. i. Inorganic salts These are not soluble in high organic solvents. Buffers containing sodium (Na + ), potassium (K + ), phosphate (PO 4 3- ) should be avoided because they are not volatile. Substitution of ammonium (NH 4 + ),acetate or formate make the buffers volatile and MS compatible. d) Column ii Proteins Proteins (e.g. from tissues, blood or serum samples) will precipitate in high (>40%) organic solvents. The precipitated proteins can clog injectors and tubing. They can also adsorb the analyte or contaminates. iii. Sample Prep Sample preparation chemicals (e.g. detergents, salts) can produce large unwanted ions. Sample cleanup needs to be LC/MS friendly. iv. Solubility Analyte solubility can be a problem. If the sample is dissolved in high organic and then injected into initial mobile phase that is low in organic, some analyte could precipitate out of solution and contaminate the injector and/or the column. These species may then re-dissolve when a higher organic content gradient is subsequently introduced. v. Injector contamination Injection of very high concentrations of sample may contaminate the injector leading to sample carryover into the next injection. This can commonly occur when looking for impurities in the sample at 0.1% level where the main sample component may be very concentrated. HPLC columns can behave like a filter. Particles, precipitated proteins, etc. can stay at the head of the column and slowly bleed off, increasing the background ions. This is known as trace enrichment. i. Column types C18 analytical or trapping columns will trap hydrophobic compounds from water and will trace enrich them. Other column packings have different polarities that can trap different classes, e.g.. polar endcapped columns will retain more polar solutes. ii. Contaminant Chromatography Compounds trapped on the head of a HPLC column may elute as distinct LC peaks or as a smear across the chromatogram. iii. Trace Enrichment The trace enriching effect amplifies the amount of contamination present in the solvents or the HPLC system _1 5 of 9

6 d) Tubing iv. Equilibration pros and cons Equilibrating a C18 HPLC column for long periods of time at high aqueous will trap more contaminants. Methods requiring high aqueous conditions should be periodically cleaned with an organic mobile phase e.g. 70:30 acetonitrile:water. Ideally, a short equilibration time between samples is best, ensuring that the eluent is diverted to waste so any contaminants from the column do not flush into the MS. i. PEEK PEEK is a plastic polymer. Very hydrophobic compounds (e.g. steroids) can stick to it and contribute to contamination when they elute. Organic solvents can remove compounds (e.g. plasticizers) from the tubing, which subsequently show on the mass spectrum. It is sometimes useful to flush new PEEK tubing through with organic and aqueous solvents prior to connection to an MS. ii. Silica e) HPLC / UPLC System Silica capillary tubing has an active ion surface. Some compounds (e.g. some proteins and peptides) will stick to silica. Substances that stick to glass will probably stick to silica. When they elute they will contribute to contamination. i. Internal components, i.e. Tubing, Check Valves, Seals HPLC systems have multiple assemblies and other items that can accumulate contamination in small dead volumes. A thorough flush of the HPLC system with clean solvents can remove contaminants from the system. Sample needles, needle seals, syringes, rheodyne components etc can all contribute to background ions. Frit filters, sample filters, etc, should be replaced periodically. ii. External Tubing The tubing that connect the outlet of the HPLC system to the MS is easily replaced and can be easily troubleshooted as a potential source of background ions. iii. Solvent bottles, filters etc Solvent bottles should be washed thoroughly in a non-detergent cleaning agent, and thoroughly rinsed with those HPLC grade solvents which are to be subsequently used as mobile phases for sample analysis prior to use. Solvent bottles filters should be replaced periodically. iv. Pumps The pump and/or autosampler modules (e.g.. Acquity BSM, and SM) should be flushed with clean solvents, ensuring all solvent pathways have been selected at least once. Injector assemblies and syringes should be primed for at least cylces. v. Wash Solutions The following solutions have been used to clean LC systems (using HPLC or better grade solvents). Mixture 1: 25% Acetonitrile 25% Methanol 25% Water 25% Isopropanol 0.2 % Formic acid _1 6 of 9

7 Mixture 2: 50% Acetonitrile 49% Water 1% Ammonium Hydroxide f) MS System The use of strongly acidic wash solution are not generally recommended for cleaning LC systems are connected to MS systems, but if used the mixture should be thoroughly washed out of the system subsequently with water. Mixture 1 has been used as a strong universal wash solution, and can be used to do a precursor wash of an LC system during installation, if required. This mixture should be thoroughly flushed out of the system with another solution, e.g. 10:90 Acetonitrile:Water. Mixture 2 has been used to reduce PEG contamination, and should also be followed by a 10:90 Acetonitirle:Water solution after use. After any wash, it is recommended that mobile phase be washed through the system to equilibrate the LC and MS at initial conditions. Mass spectrometers can be used to analyse for many different types of sample. A portion of the samples admitted to the system can become stuck or deposit in various parts of the instrument. Typically 90% of all MS contamination can be removed by cleaning the source region as described in the instrument Operators Guide. Generally there is no need to go any further inside the instrument for routine maintenance cleaning. i. ESI / APCI probe / Corona Pin The probe is where the ions are created. Therefore, any contaminants present in this region are likely to ionise and be seen in the mass spectrum. Is it good practice to rebuild the ESI probe with new components e.g. Capillary, probe tip etc, to reduce the risk of contaminant build up. When troubleshooting contaminant location, it is useful to try a replacement probe. ii. Sample Cone The sample cone is the entry point of all ions into the MS. It is good practice to clean this periodically by sonicating in a 10% formic acid solution, followed by 50:50 Methanol:Water rinse. iii. Lockspray Components MS instruments with Lockspray have additional components that can accumulate contaminants. The Reference probe should be cleaned in the same way as the main analyte probe. The baffle can be cleaned as per the sample cone. iv. Sample lines/syringes The sample introduction flow path can be prone to build up of samples/matrices/impurities in solvents etc. It is recommended that the silica infusion line/peek fittings etc be changed frequently. The sample or lockspray syringe can be cleaned with solvents and/or mobile phase appropriate to the sample types that has been used. v. Ion guides and hexapoles Most Waters instruments use either ion guides or hexapoles to transport ions from the ion source to the analyser. Usually, at least one of these should be cleaned periodically refer to the specific instrument Operator s Guide. vi. Other MS assemblies MS systems contain other assemblies that may require cleaning from time to time. Most of these are only accessible by a trained engineer 34301_1 7 of 9

8 e.g.. Pre/post-filters and collision cells on quadrupole MS instruments, transfer lens assemblies on TOF MS instruments. Details of cleaning procedures can be found in instrument Techincal Support Note documents. NOTE: It is generally NOT recommended that quadrupole assemblies be cleaned on a customer site under normal circumstances. 3. Contamination Location Strategy The following table is a strategy for locating the source of background ions. Step Test 1 Can the background ions be tuned away. E.g, are they affected by sample cone voltage, ESI capillary voltage etc 2 Using a syringe pump fitted, directly infuse a sample of the mobile phase solutions (extracted from those solutions currently in use by the HPLC) 3 Put clean mobile phase mixture through each pump of the HPLC, UPLC or CapLC to the MS with no column at a low flow rate. Background Level Changes Constant High Low Higher Lower Next Action Contamination is external to MS. E.g. HPLC system Contamination may be on internal MS component. Go to cleaning procedures. a. Solvent, water, and or acid contaminated. Try different source of solvents and acid. b. Infusion kit, bottle or MS dirty. Clean or replace. Contamination is not in solvents or acid. Go to Step 3. HPLC system is contributing contamination. Go to Step 4. Not coming from the LC system. Go to Step 5. 4 If possible, disconnect the pump from the autosampler. Pump solvent mixture directly into MS using Inlet pump without autosampler. 5 Pump mobile phase mixture through the parts of the system, adding them one at a time tubing, filter(s), column(s). High Low High Contamination is in the pump. Go to cleaning procedures. Contamination is in the autosampler. Go to cleaning procedures. The lasted added component contains the contamination Go to cleaning procedures _1 8 of 9

9 iv. Other information It is often not possible to identify the last sample or conditions that introduced contamination to an LC/MS system. However, the general lab environment may provide an indication of possible sources of background ions. For example, questions to bear in mind could be: 1. What type of lab is this? Open access: Many different sample types, usually the same mobile phase conditions. Most open access labs keep a sample record with details of previous sample types, names, concentrations, matrices etc. R&D: Generally have a few target compounds under routine analysis. Has a new analysis study been started recently? Did the research project change recently? Purification / Trace analysis: Generally have small numbers of samples, but in large quantities/concentrations. May have to inject a concentrated sample to see impurities at <1% level. 2. What samples have been analysed? Is there a sample log? Have there been any new users of the LC/MS system recently? 3. Are there any other LC or MS systems in the room? If so, what samples are they running? Under what conditions? Could a more concentrated sample have been run on this instrument by mistake? 4. Have the solvent suppliers changed recently? Some manufactures solvents are not as clean as others. Has a new batch just been started? 5. Is the API gas supply pure? If possible, replace the supply with a bottled supply to rule out a contaminated N 2 supply. 6. How are solvent bottles cleaned? Is there a communal dishwasher? Are detergents or surfactants used? 34301_1 9 of 9

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