55000 Amino Acids and Acylcarnitines from Dried Blood

MassChrom® Amino Acids and Acylcarnitines from Dried Blood - LC-MS/MS

Order no.: 55000
960 Tests
  • Acylcarnitines & free carnitine
  • Amino acids

With succinylacetone upgrade
All required reagents and isotope labelled standards included
Dried blood spot controls included
Multiple diagnoses in a single run


Description

Clinical relevance

Newborn screening aims to detect congenital metabolic disorders early on before they can cause harm to the organism. Included in these diseases are defects of amino acid metabolism such as phenylketonuria (PKU) and maple syrup urine disease (MSUD), defects of fatty acid metabolism and organic acidurias (isovaleric acidemia). If these diseases are not diagnosed, the metabolic disorders can already lead to severe irreversible harm to the newborns within their first few days of life.

Mass spectrometry is the method of choice for diagnostic testing: it enables to check for a broad range of metabolic defects in a single run. An HPLC column is not required due to the high selectivity of LC-MS/MS. This makes it possible to achieve a very precise quantification in a very short analysis time of less than 2 minutes.

 

Product advantages

  • Multiple diagnoses in a single run
  • High selectivity and sensitivity by MS/MS detection
  • Dried blood spot controls included
  • Succinylacetone upgrade available

 

This Chromsystems kit allows the very fast and semiquantitative determination of amino acids, acylcarnitines and succinylacetone (SUAC) from dried blood spot samples as part of newborn screening using tandem mass spectrometry. Sample preparation can be performed in less than 2 hours and is based on the effective extraction of the analytes from the filter card with subsequent derivatisation. 27 stable isotope labelled internal standards for calibration and measurement ensure reproducible quantification of the analytes. An upgrade set is available for the analysis of succinylacetone (see accessories). No cancerogenous hydrazine is used in this method, as is the case in many others. Furthermore, a kit that does not involve derivatisation (order no. 57000) is also available.

Chromatogram

Technical Data

Method of Analysis

LC-MS/MS

Number of Tests

960

Parameter

Acylcarnitines & free carnitine, Amino acids

Analysis Time

< 2 min

Limit of quantification

Amino acids with SUAC: 0.5-5 µmol/l
(Acyl-)carnitines: 0.05-0.5 µmol/l

Linearity

Amino acids with SUAC: 250-2500 µmol/l
(Acyl-)carnitines: 11-240 µmol/l

Recovery

Amino acids with SUAC: 51-94 %; (Acyl-)carnitines: 62-89 %

Intraassay

Amino acids with SUAC: CV = 4-10 %
(Acyl-)carnitines: CV = 4-20 %

Interassay

Amino acids with SUAC: CV = 5-9 %
(Acyl-)carnitines: CV = 8-28 %

Cutoff thresholds

Amino acids 22-743 µmol/l
Free carnitine 50-60 µmol/l
Acylcarnitines 0.1-48 µmol/l
SUAC 1.16 µmol/l

Information


Mass transitions of analytes and internal standards

Amino acids Neutral Loss Scan 102
Alanine 146 > 44
Alanine-D4 150 > 48
Aspartic acid 246 > 144
Aspartic acid-D3 249 > 147
Glutamic acid 260 > 158
Glutamic acid-D5 265 > 163
Leucine 188 > 86
Leucine-D3 191 > 89
Methionine 206 > 104
Methionine-D3 209 > 107
Phenylalanine 222 > 120
Phenylalanine-D5 227 > 125
Tyrosine 238 > 136
Tyrosine-D4 242 > 140
Valine 174 > 72
Valine-D8 182 > 80
  MRM
Arginine 231>70
Arginine-D7 238 > 77
Citrulline 232 > 113
Citrulline-D2 234 > 115
Glycine 132 > 76
Glycine-13C2-15N 135 > 79
Ornithine 189 > 70
Ornithine-D6 195 > 76
Proline 172 > 116
Proline-D7 179 > 123
SUAC 211 > 109
SUAC-13C5 216 > 114
   
(Acyl-)carnitines Parent Ion Scan 85
Carnitine 218 > 85
Carnitine-D9 227 > 85
C2-Carnitine 260 > 85
C2-Carnitine-D3 263 > 85
C3-Carnitine 274 > 85
C3-Carnitine-D3 277 > 85
C4-Carnitine 288 > 85
C4-Carnitine-D3 291 > 85
C5-Carnitine 302 > 85
C5-Carnitine-D9 311 > 85
C5DC-Carnitine 388 > 85
C5DC-Carnitine-D6 394 > 85
C6-Carnitine 316 > 85
C6-Carnitine-D3 319 > 85
C8-Carnitine 344 > 85
C8-Carnitine-D3 347 > 85
C10-Carnitine 372 > 85
C10-Carnitine-D3 375 > 85
C12-Carnitine 400 > 85
C12-Carnitine-D3 403 > 85
C14-Carnitine 428 > 85
C14-Carnitine-D3 431 > 85
C16-Carnitine 456 > 85
C16-Carnitine-D3 459 > 85
C18-Carnitine 484 > 85
C18-Carnitine-D3 487 > 85

Flow rate

20-600 µl/min

Specimen

Dried blood

Pre-analytic Treatment

Specimen

  • Take blood from the heel of the newborn onto a suitable filter card. Dry 4 hours at ambient temperature.

Time of sample drawing

  • 48th to 72nd hour of life.

Sample Preparation

Sample preparation (general)

  • Punch a 3 mm dried blood spot disk out of the filter card into a well of a microtiter plate (or a filter plate).
  • Add 200 µl of the reconstituted Internal Standard, seal with a protective sheet and agitate for 20 min at 600 rpm and room temperature.
  • Transfer the supernatant into a new microtiter plate. When using a filter plate draw through the supernatant into the well plate by centrifugation (4000 rpm, 2 minutes). Discard the filter plate.
  • Evaporate at 60 °C and 600 rpm to dryness.
  • Add 60 µl Derivatisation Reagent, seal microtiter plate with a protective sheet and incubate 15 min at 60 °C and 600 rpm.
  • Evaporate at 60 °C and 600 rpm to dryness.
  • Add 100 µl Reconstitution Buffer and agitate 1 min at 600 rpm.
  • Inject 10 µl into the LC-MS/MS system.

Sample preparation (succinylacetone)

  • Punch a 3 mm dried blood spot disk out of the filter card into a 96 well plate (or a filter plate).
  • Add 200 µl Internal Standard, seal with a protective sheet and agitate for 20 min at 600 rpm and room temperature.
  • Transfer the supernatant into a new 96 well plate. When using a filter plate draw through the supernatant into the 96 well plate by centrifugation (4000 rpm, 2 minutes).
  • Add 150 µl Internal Standard Succinylacetone to the remaining blood spot and agitate for 30 min at 60 °C.
  • Pool the supernatants and evaporate at 60 °C and 600 rpm to dryness.
  • Add 60 µl Derivatisation Reagent, seal microtiter plate with a protective sheet and incubate 15 min at 60 °C and 600 rpm.
  • Evaporate at 60 °C and 600 rpm to dryness.
  • Add 200 µl Reconstitution Buffer and agitate 1 min at 600 rpm.
  • Inject 10 µl into the LC-MS/MS system.

Sample Stability

At ambient temperature (+20 to 25 °C) or cooled (+2 to +8 °C) for up to 21 days. For longer storage the samples should be frozen with humidity excluded (below -18 °C). Avoid temperatures above 37 °C.

Injection Volume

10 µl

Autosampler rinsing solution

Rinsing Solution (55007)

MS/MS-Mode

Amino acids: Neutral Loss Scan of 102 (m/z); Acylcarnitines: Parent Ion Scan of 85 (m/z)

Additional Info

No HPLC column is required.

Please note

The freely available information on this website, in particular on the sample preparation, are not sufficient to work with our products. Please read instructions and warning notices on products and/or instruction manuals.

Kit Components

Controls

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