Acylglycine Analysis by Ultra‐Performance Liquid Chromatography‐Tandem Mass Spectrometry (UPLC‐MS/MS)

Judith A. Hobert1, Aiping Liu1, Marzia Pasquali1

1 ARUP Institute for Clinical and Experimental Pathology, University of Utah, Salt Lake City, Utah
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 17.25
DOI:  10.1002/cphg.19
Online Posting Date:  October, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Quantitative analysis of urine acylglycines has shown to be a highly sensitive and specific method with proven clinical utility for the diagnosis of several inherited metabolic disorders including: medium chain acyl‐CoA dehydrogenase deficiency, multiple acyl‐CoA dehydrogenase deficiency, short chain acyl‐CoA dehydrogenase deficiency, 3‐methylcrotonyl‐CoA carboxylase deficiency, 2‐methylbutyryl‐CoA dehydrogenase deficiency, isovaleric acidemia, propionic academia, and isobutyryl‐CoA dehydrogenase deficiency. Here, a method that is currently performed using ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC‐MS/MS) is described. © 2016 by John Wiley & Sons, Inc.

Keywords: acylglycine; fatty acid β‐oxidation; glycine N‐acylase; organic acidemia; UPLC‐MS/MS

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Urinary Acylglycine Analysis by UPLC‐MS/MS
  • Support Protocol 1: Quality Control Preparation
  • Support Protocol 2: Internal Standard (ISTD) Preparation
  • Support Protocol 3: Standard (STD)/Calibrator Preparation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: Urinary Acylglycine Analysis by UPLC‐MS/MS

  • Methanol (Fisher, cat. no. A456)
  • Standards (STDs) (see protocol 4)
  • Nitrogen source
  • Nanopure water, freshly produced
  • Quality control samples (see protocol 2)
  • Internal standards (ISTDs) (see protocol 3)
  • Urine sample
  • 3 N HCl in butanol (Regis Technologies, cat. no. 201007)
  • Ethyl acetate (VWR, cat. no. EM‐EX0241)
  • Buffer A (see recipe)
  • ∼98% formic acid (Sigma, cat. no. 94318)
  • Acetonitrile (Fisher, cat. no. A955)
  • Mobile phase A (see recipe)
  • Mobile phase B (see recipe)
  • Buffer B (see recipe)
  • Seal wash (see recipe)
  • Argon collision gas
  • Collection plates (deep‐well, 96‐well format, 1‐ml wells; Waters Corporation, cat. no. WAT058957)
  • Disposable, borosilicate glass culture test tubes, 16 × 100–mm (VWR, cat. no. 47729‐576)
  • Positive displacement pipettor
  • Repeater pipettor
  • Safe‐T‐flex caps, 16‐mm (VWR, cat. no. 60828‐766)
  • Multi‐tube shaker/vortexer (VWR, model no. VX‐2500)
  • Centrifuge
  • Evaporator for 96‐well plates (e.g., Biotage SPE Dry 96)
  • Pierceable mat cap for deep‐well plates, clear (VWR, cat. no. 62406‐512)
  • Plate rotator (Barnstead Lab‐Line model no. 2314)
  • 65ºC incubation oven
  • Acquity UPLC BEH C18 column, 1.7‐µm, 1.0 × 100–mm (Waters, cat. no. 186002346)
  • Acquity UPLC Column In‐Line Filter kit (Waters, cat. no. 205000343)
  • Acquity UPLC/Quattro Premier XE Tandem Mass Spectrometer (Waters Corporation)
  • QuantLynx and TargetLynx software (Waters Corporation)
  • Webseal, 96‐square‐well, pre‐slit, mat cap, blue (Waters Corporation, cat. no. 186000857)
NOTE: The creatinine value for urine samples is required for the calculation of acylglycine concentration. It is assumed that laboratories use standard procedures for determining creatinine concentration. The procedure for determining creatinine concentration is not described in this unit.

Support Protocol 1: Quality Control Preparation

  • Urine sample from unaffected individual or pooled normal urine samples
  • 5 mM acylglycine stock solution (see protocol 4)
  • Nitrogen source
  • 1.5‐ml microcentrifuge tubes
  • 100‐ml volumetric flasks

Support Protocol 2: Internal Standard (ISTD) Preparation

  • Internal standards (may be purchased from other suppliers):
    • [3,3,3‐d 3]Propionylglycine (d 3‐PG)
    • [d 7]Isobutyrylglycine (d 7‐IBG)
    • [4,4,4‐d 3]Butyrylglycine (d 3‐BG)
    • [d 9]2‐Methylbutyrylglycine (d 9‐2MBG)
    • [d 9]Isovalerylglycine (d 9‐IVG)
    • [5,5,5‐d 3]Valerylglycine (d 3‐VG)
    • [6,6,6‐d 3]Hexanoylglycine (d 3‐HG)
    • [d 5]Phenylpropionylglycine (d 5‐PPG)
    • [Glycine‐13C 2, 15 N]tiglylglycine (Cambridge Isotopes)
    • [Glycine‐13C 2, 15 N]3‐methylcrotonylglycine (Cambridge Isotopes)
    • [Glycine‐13C 2, 15 N]suberylglycine (Cambridge Isotopes, cat. no. CNLM‐8183)
    • Methanol
    • Nitrogen source
    • Nanopure water
    • 50‐ml volumetric flasks

Support Protocol 3: Standard (STD)/Calibrator Preparation

  • Calibrator solutions:
    • Propionylglycine (PG)
    • Isobutyrylglycine (IBG)
    • Butyrylglycine (BG)
    • 2‐Methylbutyrylglycine (2MBG)
    • Isovalerylglycine (IVG)
    • Valerylglycine (VG)
    • Tiglylglycine (TG)
    • 3‐Methylcrotonylglycine (3MCG)
    • Hexanoylglycine (HG)
    • N‐Octanoylglycine (OG)
    • Phenylpropionylglycine (PPG)
    • N‐(Cinnamoyl)glycine (CG) (Sigma‐Aldrich, cat. no. S658200)
    • Suberylglycine (SG)
  • Methanol
  • 50‐ml volumetric flasks
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Bonafé, L., Troxler, H., Kuster, T., Heizmann, C., Chamoles, N., Burlina, A., and Blau, N. 2000. Evaluation of urinary acylglycines by electrospray tandem mass spectrometry in mitochondrial energy metabolism defects and organic acidurias. Mol. Genet. Metab. 69:302‐311. doi: 10.1006/mgme.2000.2982.
  Cater, S., Midgley, J., Watson, D., and Logan, R. 1991. Measurement of urinary medium chain acyl glycines by gas chromatography‐negative ion chemical ionization mass spectrometry. J. Pharm. Biomed. Anal. 9:969‐975. doi: 10.1016/0731‐7085(91)80032‐5.
  Costa, C., Guérand, W., Struys, E., Holwerda, U., ten Brink, H., Almeida, T., Duran, M., and Jakobs, C. 2000. Quantitative analysis of urinary acylglycines for the diagnosis of β‐oxidation defects using GC‐NCI‐MS. J. Pharm. Biomed. Anal. 21:1215‐1224. doi: 10.1016/S0731‐7085(99)00235‐6.
  Fong, B., Tam, S., and Leung, K. 2012. Quantification of acylglycines in human urine by HPLC electrospray ionization‐tandem mass spectrometry and the establishment of pediatric reference interval in local Chinese. Talanta 88:193‐200. doi: 10.1016/j.talanta.2011.10.031.
  Gregersen, N., Keiding, K., and Kølvraa, S. 1979. N‐Acylglycines: Gas chromatographic mass spectrometric identification and determination in urine by selected ion monitoring. Biomed. Mass Spectrom. 6:439‐443. doi: 10.1002/bms.1200061007.
  Gregersen, N., Kølvraa, S., and Mortensen, P. 1986. Acyl‐CoA: Glycine N‐acyltransferase: In vitro studies on the glycine conjugation of straight‐ and branched‐chain acyl‐CoA esters in human liver. Biochem. Med. Metab. Biol. 35:210‐218. doi: 10.1016/0885‐4505(86)90076‐9.
  Hine, D., Hack, A., Goodman, S., and Tanaka, K. 1986. Stable isotope dilution analysis of isovalerylglycine in amniotic fluid and urine and its application for the prenatal diagnosis of isovaleric acidemia. Pediatr. Res. 20:222‐226. doi: 10.1203/00006450‐198603000‐00005.
  Kimura, M. and Yamaguchi, S. 1999. Screening for fatty acid beta oxidation disorders acylglycine analysis by electron impact ionization gas chromatography‐mass spectrometry. J. Chromatogr. B. 731:105‐110. doi: 10.1016/S0378‐4347(99)00208‐X.
  Millington, D., Kodo, N., Terada, N., Roe, D., and Chance, D. 1991. The analysis of diagnostic markers of genetic disorders in human blood and urine using tandem mass spectrometry with liquid secondary ion mass spectrometry. Int. J. Mass Spectrom. Ion Process. 111:211‐228. doi: 10.1016/0168‐1176(91)85056‐R.
  Ombrone, D., Salvatore, F., and Ruoppolo, M. 2011. Quantitative liquid chromatography coupled with tandem mass spectrometry analysis of urinary acylglycines: Application to the diagnosis of inborn errors of metabolism. Anal. Biochem. 417:122‐128. doi: 10.1016/j.ab.2011.05.042.
  Pasquali, M. and Longo, N. 2012. Newborn Screening and Inborn Errors of Metabolism. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 5th Edition. p. 2055. (C. Burtis, E. Ashwood, and D. Bruns, eds.) Elsevier, Saunders, St. Louis, Missouri. ISBN: 978‐1‐4160‐6164‐9.
  Shigematsu, Y., Hata, I., and Tanaka, Y. 2007. Stable‐isotope dilution measurement of isovalerylglycine by tandem mass spectrometry in newborn screening for isovaleric acidemia. Clin. Chim. Acta 386:82‐86. doi: 10.1016/j.cca.2007.08.003.
  Stanislaus, A. and Li, L. 2010. A method for comprehensive analysis of urinary acylglycines by using ultra‐performance liquid chromatography quadrupole linear ion trap mass spectrometry. J. Am. Soc. Mass Spectrom. 21:2105‐2116. doi: 10.1016/j.jasms.2010.09.004.
  Stanislaus, A., Guo, K., and Li, L. 2012. Development of an isotope labeling ultra‐high performance liquid chromatography mass spectrometric method for quantification of acylglycines in human urine. Anal. Chim. Acta 750:161‐172. doi: 10.1016/j.aca.2012.05.006.
  Van den Berg, H. and Hommes, F. 1975. Thin‐layer chromatographic detection of some N‐acylglycine conjugates in urine. J. Chromatogr. A 104:217‐222. doi: 10.1016/S0021‐9673(01)85517‐5.
PDF or HTML at Wiley Online Library