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Internal standards

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Internal standards are reference compounds used in analytical chemistry to aid in the quantification of target analytes. They are added to samples in a known concentration and their signal response is used to determine the concentration of the analytes of interest.

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Lab products found in correlation

4000 qtrap, by AB Sciex (2 mentions) Isopropyl alcohol, by Avantor (2 mentions) Low retention microcentrifuge tube, by Thermo Fisher Scientific (2 mentions) Serological pipettes, by Thermo Fisher Scientific (2 mentions) Plastic pipette tips, by USA Scientific (2 mentions) Acetic acid, by Thermo Fisher Scientific (2 mentions) Acetonitrile, by Thermo Fisher Scientific (2 mentions) Methanol, by Thermo Fisher Scientific (2 mentions) Chloroform, by Thermo Fisher Scientific (2 mentions) Pyrex glass culture tubes, by Corning (2 mentions) Methyl tert butyl ether (mtbe), by Avantor (2 mentions) Dichloromethane, by Merck Group (1 mentions) Bovine heart extract, by Avanti Polar Lipids (1 mentions) Ascentis express c18 column, by Merck Group (1 mentions) Qtrap 5500 tandem mass spectrometer, by AB Sciex (1 mentions) Analyst software v1, by AB Sciex (1 mentions) Multiquant software 3, by AB Sciex (1 mentions) Fty720, by Cayman Chemical (1 mentions)

14 protocols using internal standards

1

Internal Standard Addition for Lipid Analysis

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Internal standards were purchased from Avanti Polar Lipids (Alabaster, AL) and added to samples in 20 μl ethanol:methanol:water (7:2:1) as a cocktail of 500 pmol each. The HPLC grade solvents were obtained from VWR (West Chester, PA).
Nagahashi M., Yamada A., Aoyagi T., Allegood J., Wakai T., Spiegel S, & Takabe K. (2016). Sphingosine-1-phosphate in the lymphatic fluid determined by novel methods. Heliyon, 2(12), e00219.
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2

Sphingolipid Quantification in Perfused Lungs

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Serum and saline perfused lung tissues were collected from the mice. After the addition of internal standards (0.5 nmol each; Avanti Polar Lipids, Alabaster, Alabama, USA) the lipids were extracted and sphingolipids were quantified by LC-ESI-MS/MS (4000 QTRAP, AB Sciex, Framingham, Massachusetts, USA) as described previously46 (link).
van der Weyden L., Arends M.J., Campbell A.D., Bald T., Wardle-Jones H., Griggs N., Velasco-Herrera M.D., Tüting T., Sansom O.J., Karp N.A., Clare S., Gleeson D., Ryder E., Galli A., Tuck E., Cambridge E.L., Voet T., Macaulay I.C., Wong K., Spiegel S., Speak A.O, & Adams D.J. (2017). Genome-wide in vivo screen identifies novel host regulators of metastatic colonization. Nature, 541(7636), 233-236.
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3

Sphingolipid Quantification in Mice Skin

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Internal standards (0.5 nm each; Avanti Polar Lipids, Alabaster, AL, USA) were added to samples, lipids were extracted from dorsal skin and tail skin (separated into the dermis and epidermis) of young (9 weeks) and old (32 weeks) Acer1+/+ and Acer1−/− male mice, and sphingolipids were quantified by LC–ESI–MS/MS (4000 QTRAP, AB Sciex, Framingham, MA, USA), as described previously 9.
Liakath‐Ali K., Vancollie V.E., Lelliott C.J., Speak A.O., Lafont D., Protheroe H.J., Ingvorsen C., Galli A., Green A., Gleeson D., Ryder E., Glover L., Vizcay‐Barrena G., Karp N.A., Arends M.J., Brenn T., Spiegel S., Adams D.J., Watt F.M, & van der Weyden L. (2016). Alkaline ceramidase 1 is essential for mammalian skin homeostasis and regulating whole‐body energy expenditure. The Journal of Pathology, 239(3), 374-383.
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4

Sphingolipid Quantification by LC-ESIMS/MS

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Internal standards were purchased from Avanti Polar Lipids (Alabaster, AL) and added to samples in 20 μl ethanol:methanol:water (7:2:1) as a cocktail of 500 pmol each. The HPLC grade solvents were obtained from VWR (West Chester, PA). Lipids were extracted from tissue samples and sphingolipids were quantified by liquid chromatography, electrospray ionization-tandem mass spectrometry (LC-ESIMS/MS, 4000 QTRAP, ABI) as described previously at Virginia Commonwealth University Lipidomics Core [13 (link), 23 (link)].
Nagahashi M., Tsuchida J., Moro K., Hasegawa M., Tatsuda K., Woelfel I.A., Takabe K, & Wakai T. (2016). High levels of sphingolipids in human breast cancer. The Journal of surgical research, 204(2), 435-444.
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5

Quantification of Deuterated Phospholipids in Plasma

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Internal standards (Avanti Polar Lipids) of 10 nmol of dimyristoyl-PC (PC14:0/14:0), 1 nmol of lysophosphatidylcholine (LPC)17:0, and 50 pmol of D4-choline were added to 100 μl of plasma. The phospholipid fraction was extracted by chloroform:methanol:water (v/v, 2:2:1) (12 (link)). The lower chloroform-rich layer was dried under nitrogen gas at 37°C and was directly infused through MS with an electrospray interface after dissolving with methanol:butanol:water:25% NH4OH (6:2:1.6:0.4, v/v) at a rate of 8 μl/min. Collision-induced decomposition resulted in a protonated phosphocholine head group with m/z of +184 for PC, m/z of +187 for the incorporation of single-deuterated methyl-PC group, m/z of +190 for double-deuterated methyl-PC groups, and m/z of +193 for triple-deuterated methyl-PC groups. Application of specific precursor ion scans (P184, P187, P190, and P193) resulted in quantification of endogenous and one, two, and three deuterated methyl-PC molecular species compositions and concentrations and, hence, enrichment.
Dushianthan A., Cusack R., Grocott M.P, & Postle A.D. (2018). Abnormal liver phosphatidylcholine synthesis revealed in patients with acute respiratory distress syndrome. Journal of Lipid Research, 59(6), 1034-1045.
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6

Quantification of Circulating S1P and Inflammatory Cytokines

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Circulating S1P concentration was quantified using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESIMS/MC) at the Virginia Commonwealth University Lipidomics Core as previously described [13 (link), 14 (link)]. Briefly, this consisted of internal standards from Avanti Polar Lipids (Alabaster, AL), lipid extraction in 20 μL aliquot of ethanol : methanol : water (7 : 2 : 1), and analysis for S1P using LC-ESIMS/MC.
Additionally, levels of various circulating inflammatory cytokines were measured from baseline serum samples with enzyme-linked immunosorbent assays. The cytokines measured included interleukin- (IL-) 1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, G-CSF, GM-CSF, interferon gamma (IFN-γ), MCP-1, MIP-1, and tumor necrosis factor alpha (TNF-α).
Ramanathan R., Raza A., Sturgill J., Lyon D., Young J., Hait N.C, & Takabe K. (2017). Paradoxical Association of Postoperative Plasma Sphingosine-1-Phosphate with Breast Cancer Aggressiveness and Chemotherapy. Mediators of Inflammation, 2017, 5984819.
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7

Quantitative Lipid Profiling from Plasma

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Mass spectrometry–based lipid analysis was performed as described by Surma and colleagues [18 (link)]. For lipid extraction, an equivalent of 1 μL of undiluted plasma was used, and plasma lipids were extracted with methyl tert-butyl ether/methanol (7:2, V:V) [54 (link)]. Internal standards (Avanti Polar Lipids, Birmingham, AL) were premixed with the organic solvents mixture. The Internal standards included known amounts of: cholesterol D6 (Chol), cholesterol ester 20:0 (CE), ceramide 18:1;2/17:0 (Cer), diacylglycerol 17:0/17:0 (DAG), phosphatidylcholine 17:0/17:0 (PC), phosphatidylethanolamine 17:0/17:0 (PE), lysophosphatidylcholine 12:0 (LPC), lysophosphatidylethanolamine 17:1 (LPE), triacylglycerol 17:0/17:0/17:0 (TAG), and sphingomyelin 18:1;2/12:0 (SM). After extraction, the organic phase was transferred to an infusion plate and dried in a speed vacuum concentrator. Dried extract was resuspended in 7.5 mM ammonium acetate in chloroform/methanol/propanol (1:2:4, V:V:V). All liquid handling steps were performed using Hamilton Robotics STARlet (Hamilton Robotics, Reno, NV) with the Anti Droplet Control feature for organic solvents pipetting. Chemicals and solvents of HPLC/LC-MS analytical grade were used (Merck, Darmstadt, Germany).
Gerl M.J., Klose C., Surma M.A., Fernandez C., Melander O., Männistö S., Borodulin K., Havulinna A.S., Salomaa V., Ikonen E., Cannistraci C.V, & Simons K. (2019). Machine learning of human plasma lipidomes for obesity estimation in a large population cohort. PLoS Biology, 17(10), e3000443.
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8

Lipid Standards for Mass Spectrometry

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Dichloromethane (Sigma-Aldrich), methanol and ammonium acetate (Fluka), and water (Burdick and Jackson) were LC-MS grade. Teflon coated caps and glass tubes were from VWR. 12 mm x 32 mm amber vials were from Thermo Scientific and 9 mm threaded caps were from Wheaton Inc. Internal standards (Avanti Polar Lipids, Inc.) were used for monitoring lipid class recoveries for: TAGs, D5-TAG (1,3 dipentadecanoyl-2-(9z-octadecanoyl)-sn-glycerol 2H5, Fw 810.34), GPLs, PC 17:0/14:1 (1-heptadecanoyl-2-(9z-tetradecanoyl)-sn-glycerol-phospholcholine, Fw 718.00), sphingosines, SM 18:1;2/17:0 (n-heptadecanoyl-D-erythro-sphingosylphosphocholine, Fw 717.07). Bovine heart extract (Avanti Polar Lipids, Inc.) at 0.25-2.5 ng/ml in methanol/Dichloromethane/ammonium acetate infusion solvent was used for mass (MS and MS/MS) calibration.
Liaw L., Prudovsky I., Koza R.A., Anunciado-Koza R.V., Siviski M.E., Lindner V., Friesel R.E., Rosen C.J., Baker P.R., Simons B, & Vary C.P. (2016). Lipid profiling of in vitro cell models of adipogenic differentiation: relationships with mouse adipose tissues. Journal of cellular biochemistry, 117(9), 2182-2193.
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9

Lipidomic Analysis of Sphingolipids

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For lipidomic analysis, internal standards procured from Avanti Polar Lipids (Alabaster, AL) were added to the samples in 10 μl ethanol: methanol: water (7:2:1) cocktail at 250 pmol concentration each. Samples were disrupted using ultra-sonicator (30 secs at room temperature), followed by overnight incubation at 48°C. Further preparatory steps included addition of KOH (1M) solubilized in methanol, followed by a short sonication step, and thereafter incubation for 2 h at 37°C in a shaking water bath to cleave potentially interfering glycerolipids. Samples were neutralized followed by centrifugation to collect the supernatant. The extracts were Speed Vac dried and reconstituted in starting mobile phase solvent for LC-MS/MS (Liquid Chromatography with tandem Mass Spectrometry) analysis. Samples were further sonicated for 15 seconds and centrifuged for 5 min to collect the clear supernatant which was then transferred to the auto injector vial for analysis. LC-MS/MS analyses were performed for sphingoid bases, sphingoid base-1-phosphates, and complex sphingolipids. Separation of compounds was carried out by reverse phase LC using a Supelco 2.1 (i.d.) × 50 mm Ascentis Express C18 column (Sigma, St. Louis, MO) and a binary solvent system at 35°C. Prior to the injection of each sample, column was equilibrated for 0.5 min with a solvent mixture of 95% mobile phase.
Singh D.P., Kaur G., Bagam P., Pinkston R, & Batra S. (2018). Membrane microdomains regulate NLRP10 and NLRP12 dependent signalling in A549 cells challenged with Cigarette Smoke Extract. Archives of toxicology, 92(5), 1767-1783.
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10

Lipid Extraction and Ionization Protocol

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All solvents, reagents, and lipid standards were used in the highest available purity. Internal standards were acquired from Avanti Polar Lipids (Avanti Polar Lipids, Alabama, USA).
Storage solution was created by combining chloroform, methanol, and water (20/10/1.5; v/v/v). ESI spray mixture was created by combining chloroform, methanol with added 0.1% (wt/v) ammonia acetate and 2-propanol (1:2:4; v/v/v).
Lunding L.P., Krause D., Stichtenoth G., Stamme C., Lauterbach N., Hegermann J., Ochs M., Schuster B., Sedlacek R., Saftig P., Schwudke D., Wegmann M, & Damme M. (2021). LAMP3 deficiency affects surfactant homeostasis in mice. PLoS Genetics, 17(6), e1009619.
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