Homocysteine in Serum/Plasma - HPLC

Order No.: 45000, for 200 tests
Parameters:
Homocysteine

Fast sample preparation
Short analysis time
Column life-time more than 2000 runs
Suitable for all blood collection systems

Homocysteine

Clinical relevance

Homocysteine (Hcy) is a non-proteinogenic amino acid, synthesized during the demethylation of the amino acid methionine. Some enzymes are involved in this process, which require cofactors such as vitamin B6 and vitamin B12. A deficiency of these cofactors can lead to an increase in the Hcy concentration, but inherited genetic mutations may also be the cause.

Even moderately increased Hcy levels are a risk factor for cardiovascular diseases, arteriosclerosis and coronary heart diseases. Increased Hcy levels are also detected in the case of homocystinuria, an inherited genetic disorder in which the enzymes required for metabolism are not present or at levels that are too low. In this case, already newborns have high levels of Hcy, however, the disease is treatable if detected in time. Screening of newborns (NBS) for homocystinuria has therefore become mandatory in some European countries such as Ireland. In Germany it is only performed as part of more intensive screening or in cases where the disease is suspected.

 

Product advantages

  • Fast sample preparation
  • Short analysis time
  • Column lifetime of more than 2000 runs
  • For all blood collection systems

 

This reagent kit allows the simple and reliable determination of total homocysteine in serum/plasma. Sample preparation is simply a reduction step for releasing homocysteine from its protein binding, followed by precipitation and subsequent precolumn derivatisation. Chromatographic separation is performed on an isocratic HPLC system with fluorescence detection. The optimised reagents ensure the accurate and reliable quantification of total homocysteine for all blood collection systems within a brief analysis period of less than 6 minutes.

More Information
Number of Tests 200
Limit of quantification 0.4 µmol/l
Linearity up to 400 µmol/l
Recovery 96–99 %
Intraassay

CV = 0.9–2.2 %

Interassay

CV = 2.9–3.1 %

Analysis Time 5.5 min
Sample Preparation
  • To 100 µl plasma/serum add 25 µl Internal Standard and 75 µl Reduction Reagent.
  • Shake for 2 s (vortex).
  • Incubate 10 min at ambient temperature.
  • Add 100 µl Precipitation Reagent.
  • Shake for 30 s (vortex).
  • Centrifuge 5 to 7 min at 9000 x g.
  • Pipette 100 µl derivatisation mix into a new light protected reaction vial.
  • Add 50 µl supernatant, mix well.
  • Incubate 10 min at +50 to +55 °C.
  • Cool down immediately.
  • Inject 20 to 50 µl into the HPLC system.
Sample Stability The prepared samples are stable at ambient temperature up to 20 h, at +2 to +8 °C for 5 days and below -18 °C up to 3 weeks.
Specimen Serum/Plasma
Pre-analytic Treatment

Blood should be taken from the fasted patient. If no special homocysteine blood collecting systems are used, plasma/serum should be prepared within 45 min after drawing the sample. Homocystein in plasma is stable at room temperature for 24 h, at +4 °C up to 1 week and below -18 °C several months.

Injection Volume 20–50 µl
Flow rate 1.3–1.7 ml/min
Column temperature ambient (~ 25 °C)
Wavelength

EX 385 nm
EM 515 nm

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.
Method of Analysis HPLC
Parameter Homocysteine
Complete Specification
Linearity: up to 400 µmol/l
Limit of quantification: 1.0 µmol/l
Intra-assay:
< 3 %
Inter-assay: < 4 %
Recovery: 91 %
Run time: 5.5 min

HPLC parameters

For the analysis of homocysteine any isocratic HPLC system with fluorescence detector is suitable.

Injection volume: 25 µl
Flow rate: 1.3 to 1.7 ml/min
Wavelengths: EX 385 nm
EM 515 nm
Column temperature: ambient (~25 °C)
Additional Info Any isocratic HPLC system with fluorescence detector is suitable.
  1. 39001 HPLC mobile phase homocysteine plasma serum
    Mobile Phase
    Order no.: 39001
    Homocysteine in Serum/Plasma - HPLC
    Details
  2. 39001 HPLC mobile phase homocysteine plasma serum
    Mobile Phase, 10 x 1000 ml
    Order no.: 39002
    Homocysteine in Serum/Plasma - HPLC
    Details
  3. 39004 HPLC plasma calibration reagent homocysteine
    Homocysteine Plasma Calibration Standard
    Order no.: 39004
    Homocysteine in Serum/Plasma - HPLC
    Details
  4. 45099 HPLC internal standard homocysteine plasma serum
    Internal Standard
    Order no.: 45099
    Homocysteine in Serum/Plasma - HPLC
    Details
  5. 45006 HPLC derivatisation reagent 1 homocysteine plasma serum
    Derivatisation Reagent 1
    Order no.: 45006
    Homocysteine in Serum/Plasma - HPLC
    Details
  6. 45007 HPLC derivatisation reagent 2 homocysteine plasma serum
    Derivatisation Reagent 2
    Order no.: 45007
    Homocysteine in Serum/Plasma - HPLC
    Details
  7. 45088 HPLC reduction reagent homocysteine plasma serum
    Reduction Reagent
    Order no.: 45088
    Homocysteine in Serum/Plasma - HPLC
    Details
  8. Reaction Vials, 1.5 ml
    Reaction Vials, 1.5 ml
    Order no.: 33005
    Amber colour, light protected
    Details
  9. 45003 HPLC precipitation reagent homocysteine plasma serum
    Precipitation Reagent
    Order no.: 45003
    Homocysteine in Serum/Plasma - HPLC
    Details
  1. 39004 HPLC plasma calibration reagent homocysteine
    Homocysteine Plasma Calibration Standard
    Order no.: 39004
    Homocysteine in Serum/Plasma - HPLC
    Details
  1. 0071 0072 0073 HPLC homocysteine plasma controls
    Homocysteine Plasma Controls
    Order no.: 0071/0072/0073
    Details
  1. PEEK Prefilter Housing
    PEEK Prefilter Housing
    Order no.: 15010
    Details
  2. 39100 HPLC column homocysteine plasma serum
    HPLC column
    Order no.: 39100
    Homocysteine in Serum/Plasma
    Details
  3. PEEK-encased Prefilter, 5 µm
    Details
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