Lidstrom:in vitro Pathway Assay: Sample Analysis via UPLC-MS/MS - Revision history

Janet B. Matsen: /* Background */

Janet B. Matsen — Mon, 07 Jan 2013 14:20:52 GMT

Background

←Older revision		Revision as of 14:20, 7 January 2013
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	** "Separations of complex mixtures normally require the elution of analytes from a buffered aqueous to an organic mobile phase composition (e.g., 5–100% organic). Thus, analytes are eluted in the order of their increasing polarity.The separation of very polar and/or ionic compoundsgenerally requires the use of a highly aqueous eluent composition (>95%) to provide some retention. Such a hydrophilic environment on the highly hydrophobic C18 stationary phase can lead to a loss of retention, increased peak tailing, and irreproducible results. This de-wetting effect (often incorrectly referred to as phase- collapse) (49) can be significantly reduced with the application ofLC columns with specially designed stationary phases (using polar embedded groups or polar end-capping, such as Aquasil/AQ columns)" - [http://www.ncbi.nlm.nih.gov/pubmed/22639216 Mass spectrometry-based microbial metabolomics] 2012 (Baidoo, Benke & Keasling)		** "Separations of complex mixtures normally require the elution of analytes from a buffered aqueous to an organic mobile phase composition (e.g., 5–100% organic). Thus, analytes are eluted in the order of their increasing polarity.The separation of very polar and/or ionic compoundsgenerally requires the use of a highly aqueous eluent composition (>95%) to provide some retention. Such a hydrophilic environment on the highly hydrophobic C18 stationary phase can lead to a loss of retention, increased peak tailing, and irreproducible results. This de-wetting effect (often incorrectly referred to as phase- collapse) (49) can be significantly reduced with the application ofLC columns with specially designed stationary phases (using polar embedded groups or polar end-capping, such as Aquasil/AQ columns)" - [http://www.ncbi.nlm.nih.gov/pubmed/22639216 Mass spectrometry-based microbial metabolomics] 2012 (Baidoo, Benke & Keasling)
	*** This is a "reverse phase" (hydrophobic) method		*** This is a "reverse phase" (hydrophobic) method
		+	=== Ion Pairing ===
		+	* Our method is ion pairing. (Tributylamine in buffer C)
		+	** "The application of ion-pairing reagents in RP LC improves the to separations of these types of compounds. However, the addition of these ion- pairing (IP) reagents is often disadvantageous for MS detection (due to ion-suppression and the possibility of contamination after prolonged used)." - [http://www.ncbi.nlm.nih.gov/pubmed/22639216 Mass spectrometry-based microbial metabolomics] 2012
		+	** ion pairing: for when analytes include basic or ionic compounds that are difficult to chromatograph by reverse-phase LC. In IPC, the mobile phase contains an ion-pair reagent that attaches to the stationary phase and creates a charged surface. The sample ion exchanges with the counter ion of the ion-pair reagent thus increasing interaction with the column resulting in greater retention of the sample.

	== References ==		== References ==

Janet B. Matsen: /* References */

Janet B. Matsen — Mon, 07 Jan 2013 14:15:58 GMT

References

←Older revision		Revision as of 14:15, 7 January 2013
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	Accela HPLC Protocol		Accela HPLC Protocol
		+
		+	== Background ==
		+	=== Column & Buffer ===
		+	*We use a C18 column, and 5% methanol in our buffer.
		+	** "Separations of complex mixtures normally require the elution of analytes from a buffered aqueous to an organic mobile phase composition (e.g., 5–100% organic). Thus, analytes are eluted in the order of their increasing polarity.The separation of very polar and/or ionic compoundsgenerally requires the use of a highly aqueous eluent composition (>95%) to provide some retention. Such a hydrophilic environment on the highly hydrophobic C18 stationary phase can lead to a loss of retention, increased peak tailing, and irreproducible results. This de-wetting effect (often incorrectly referred to as phase- collapse) (49) can be significantly reduced with the application ofLC columns with specially designed stationary phases (using polar embedded groups or polar end-capping, such as Aquasil/AQ columns)" - [http://www.ncbi.nlm.nih.gov/pubmed/22639216 Mass spectrometry-based microbial metabolomics] 2012 (Baidoo, Benke & Keasling)
		+	*** This is a "reverse phase" (hydrophobic) method

	== References ==		== References ==

Janet B. Matsen: /* Columns: */

Janet B. Matsen — Mon, 07 Jan 2013 14:08:31 GMT

Columns:

←Older revision		Revision as of 14:08, 7 January 2013
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	===Columns:===		===Columns:===
	*Waters Acquity C18 Column. Bigger blue box.		*Waters Acquity C18 Column. Bigger blue box.
-	*Also tried,	+	*Also tried:
	**Hypersil Gold AX, 100 x 2.1 mm, Thermo-scientific		**Hypersil Gold AX, 100 x 2.1 mm, Thermo-scientific
	**Hypersil Gold aQ, 100 x 2.1 mm, Thermo-scientific		**Hypersil Gold aQ, 100 x 2.1 mm, Thermo-scientific

Janet B. Matsen: /* References */

Janet B. Matsen — Wed, 26 Dec 2012 18:18:39 GMT

References

←Older revision		Revision as of 18:18, 26 December 2012
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	== References ==		== References ==
-	* [http://www.ncbi.nlm.nih.gov/pubmed/20433152 Ultrahigh performance liquid chromatography-tandem mass spectrometry method for fast and robust quantification of anionic and aromatic metabolites], 2010. This is the protocol Amanda & Justin used to get ours going.	+	* [http://www.ncbi.nlm.nih.gov/pubmed/20433152 Ultrahigh performance liquid chromatography-tandem mass spectrometry method for fast and robust quantification of anionic and aromatic metabolites], 2010. This is the protocol Amanda & Justin used to get ours going.
		+	** Their LC protocol:
		+	***“Separation of compounds was achieved by an ion pairing-reverse phase method developed for ultrahigh performance systems based on previously published high pressure methods15,17 and eventually implemented on a Waters Acquity UPLC (Waters Corporation, Milford, MA, United States) using a Waters Acquity T3 end-capped reverse phase column with dimensions 150 mm × 2.1 mm × 1.8 µm (Waters Corporation, Milford, MA, United States) temperature-controlled at 40 °C. A gradient of mobile phases A (10 mM tributylamine, 15 mM acetic acid, 5% (v/v) methanol) and B (2-propanol) was used to separate metabolites (Table 1). The injection volume was 10 µL with full loop injection. The column was equilibrated for 4 min (4.4 column volumes) before each injection. “
		+	** Their MS protocol:
		+	***“Selective and sensitive detection of compounds was achieved by coupling the liquid chromatograph to a Thermo TSQ Quantum Ultra triple quadrupole instrument (Thermo Fisher Scientific, Waltham, MA, United States) using a heated electrospray ionization source (Thermo Fisher Scientific, Waltham, MA, United States). The mass spectrometer was operated in negative mode with multiple reaction monitoring. Electrospray ionization parameters were optimized for 25% 2-pro- panol at a flow rate of 0.4 mL/min and used for the entire gradient: spray voltage 2500 V, sheath gas pressure 80 arbitrary units, aux gas pressure 50 arbitrary units, ion sweep gas pressure 5 arbitrary units, capillary temperature 380 °C, spray temperature 400 °C. Tube lens voltage, collision energy, and fragment ions were optimized individually for all compounds (Table 2). Ion optics were set to 0.5 amu Q1 resolution, 0.5 amu Q3 resolution, 0.01 amu scan width, and 10 ms dwell time. SRM scans were organized in 11 time periods so that each compound could be detected for more than 1 min before and after expected retention time and a scan frequency of at least 2 Hz was maintained. Data Analysis. Both acquisition and peak integration were performed with Xcalibur software version 2.07 SP1 (Thermo Fisher Scientific, Waltham, MA, United States) and in-house integration software (Begemann and Zamboni, unpublished). The criteria for quantifiability of a standard compound were that a minimum of 4 neighboring dilution steps showed a linear cor- relation between the logarithm of the concentration and the logarithm of the peak area with a correlation coefficient greater than 0.99. Additionally, the peak area of the lower limit of the linear range had to be larger than the peak area found in water."

	==BEFORE SETUP NOTES==		==BEFORE SETUP NOTES==

Janet B. Matsen at 17:32, 26 December 2012

Janet B. Matsen — Wed, 26 Dec 2012 17:32:07 GMT

←Older revision		Revision as of 17:32, 26 December 2012
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	Back to [[Lidstrom:Protocols\|Lidstrom Protocols]]		Back to [[Lidstrom:Protocols\|Lidstrom Protocols]]

-	Accela HPLC Protocol {Amanda ~~adapted this from someone else's notes}~~	+	Accela HPLC Protocol
		+
		+	== References ==
		+	* [http://www.ncbi.nlm.nih.gov/pubmed/20433152 Ultrahigh performance liquid chromatography-tandem mass spectrometry method for fast and robust quantification of anionic and aromatic metabolites], 2010. This is the protocol Amanda & Justin used to get ours going.

	==BEFORE SETUP NOTES==		==BEFORE SETUP NOTES==

Janet B. Matsen: /* Our Buffers: */

Janet B. Matsen — Wed, 26 Dec 2012 17:30:33 GMT

Our Buffers:

←Older revision		Revision as of 17:30, 26 December 2012
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	** for 10L: 2.38 mg/L tributylamine, 0.857 mg/L acetic acid, 5% methanol.		** for 10L: 2.38 mg/L tributylamine, 0.857 mg/L acetic acid, 5% methanol.
	** for 10L: 9.468L dH2O + 23.8 mL tributylamine + 8.57 mL acetic acid + 0.5 L methanol		** for 10L: 9.468L dH2O + 23.8 mL tributylamine + 8.57 mL acetic acid + 0.5 L methanol
		+	** tributylamine = ion pairing reagent
	* do you have enough buffer C to do your run? (calculate it)		* do you have enough buffer C to do your run? (calculate it)
	**(7/8) = estimate of fraction that is yellow (buffer C) (this is conservative)		**(7/8) = estimate of fraction that is yellow (buffer C) (this is conservative)

Amanda L Smith: /* Obtaining Data */

Amanda L Smith — Wed, 12 Sep 2012 02:03:16 GMT

Obtaining Data

←Older revision		Revision as of 02:03, 12 September 2012
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	== Obtaining Data ==		== Obtaining Data ==
-	* it forces you to save before exiting	+	* the program forces you to save before exiting
	* to download data, click on quantitate screen, then File - Export -		* to download data, click on quantitate screen, then File - Export -
	** settings: one file for all components. arrangement = Quan Results Grid. You can change it to comma separation.		** settings: one file for all components. arrangement = Quan Results Grid. You can change it to comma separation.

Amanda L Smith: /* Finishing with Equipment */

Amanda L Smith — Wed, 12 Sep 2012 01:53:25 GMT

Finishing with Equipment

←Older revision		Revision as of 01:53, 12 September 2012
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	== Finishing with Equipment ==		== Finishing with Equipment ==
	* flush column? Leave it on?		* flush column? Leave it on?
		+	** next person should flush it and remove it. If it is going to be a while before anyone uses it again, you can flush your column and store it. ?? Can you leave it without a column?? better ask first.
	* Leave liquid N2 on		* Leave liquid N2 on

Amanda L Smith: /* Misc */

Amanda L Smith — Wed, 12 Sep 2012 01:52:20 GMT

Misc

←Older revision		Revision as of 01:52, 12 September 2012
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	== Misc ==		== Misc ==
	* Do we need to order more liquid N2 tanks or does someone take care of it?		* Do we need to order more liquid N2 tanks or does someone take care of it?
		+	** Tell Adam

	==(?) SOFTWARE SETUP==		==(?) SOFTWARE SETUP==

Amanda L Smith: /* Obtaining Data */

Amanda L Smith — Wed, 12 Sep 2012 01:51:57 GMT

Obtaining Data

←Older revision		Revision as of 01:51, 12 September 2012
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	== Obtaining Data ==		== Obtaining Data ==
-	* ??	+	* it forces you to save before exiting
		+	* to download data, click on quantitate screen, then File - Export -
		+	** settings: one file for all components. arrangement = Quan Results Grid. You can change it to comma separation.

	== Finishing with Equipment ==		== Finishing with Equipment ==