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Quantitative Determination of Phenylketonuria

 Quantitative Determination of Phenylketonuria
Zivak PKU HPLC-UV Analysis Kit was developed for sensitive, rapid and accurate quantitative detection of Phenylketonuria in human dried blood spot & serum samples. Main methods and procedures that have been selected are based on EN ISO 13485 and 98/79/EC.

Phenylketonuria (PKU) is an autosomal recessive genetic disorder characterized by a deficiency in the enzyme phenylalanine hydroxylase (PAH). This enzyme is necessary to metabolise the amino acid phenylalanine to the amino acid tyrosine. When PAH is deficient, phenylalanine accumulates on the brain tissue and is converted into phenyl pyruvate (also known as phenyl ketone), which is detected in the urine. PAH is found on chromosome number 12. If left untreated, this condition can cause problems with brain development, leading to progressive mental retardation and seizures. However, PKU can be controlled by a special diet with low in phenylalanine and high in tyrosine can be a very effective treatment. There is no cure. The disorder is only detectable after irreversible damage is done hence early diagnosis is crucial. Most babies in developed countries are screened for PKU soon after birth. PKU is usually detected using the HPLC test, but some clinics still use the Guthrie test, as a part of national biochemical screening programs.

 Quantitative Determination of Phenylketonuria 0
HPLC-UV Parameters (for serum samples)

Device

Zivak ASP-100 HPLC-UV/VIS System

Column

Zivak PKU Serum HPLC Analytical Column

Injection Volume

20 µL

Pump Program

00:00 min                  100% A

09:00 min                  100% A

Flow

0.50 mL/min

Column Oven

30 °C

Detector

UV

Wavelength

214 nm

HPLC-UV Parameters (for dried blood samples)

Device

Zivak ASP-100 HPLC-UV/VIS System

Column

Zivak PKU Dried Blood Spots HPLC Analytical Column

Injection Volume

20 µL

Pump Program

00:00 min                  100% A

13:00 min                  100% A

Flow

0.50 mL/min

Column Oven

30 °C

Detector

UV

Wavelength

214 nm

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Analytical Performance

No

Analyte

LOD (µmol/L)

LOQ (µmol/L)

Accuracy (%)

Intra-Assay Precision (%CV)

Inter-Assay Precision

(%CV)

Linearity

(R2)

1

Phenylalanine (PHE)

0.35

1.05

98,4

1.4

3.2

0.997

2

Tryptophan (TRP)

0.20

0.60

97,1

1.3

3.1

0.996

3

Tyrosine (TYR)

0.45

1.35

99,5

1.4

3.2

0.998

 

 

 

 

 

 

 

 

Storage & Stability:

  • The kit can be shipped at room temperature.
  • After arrival, calibrator and controls, R1 can be stored at 2-8 °C.
  • MP, WB can be stored at room temperature.
  • All components are guaranteed until expiry date when stored at recommended temperatures and used as described in these instructions.

Preparation of Calibrators & Controls (Serum):

Add 3 ml of deionised water to plasma calibrator/control and dissolve.

After preparation, calibrators and controls must be aliquoted into 75 µl volumes and stored at -20 °C.

 
 

Sample Pre-treatment (Serum)

Take 50 mL of plasma calibrator into a 2 ml preparation tube.

 

Add 300 mL of R1. Mix by vortex for 5 seconds and incubate for 15 minutes at 4 °C.

 

Centrifuge at 5.000 x rpm for 1 minute.

 

Take 200 µl upper liquid into autosampler vial.

 

Inject 20 mL to the HPLC-UV system.

Note: The prepared sample is stable at 2-8°C for 24 hours.
 
 

Sample Pre-treatment (Dried Blood Spot)

Take dried blood spot sample in 3.5 mm diameter by a puncher into a 2 ml preparation tube. 

 

Add 300 mL of R1 to each sample. Mix by vortex for 5 secs. Incubate for 30 minutes at 37 °C.

 

Centrifuge at 12.000 x rpm for 1 minute.

 

Take 200 µl of upper liquid into autosampler vial.

 

Inject 20 mL to the HPLC-UV system.

Note: The prepared sample is stable at 2-8°C for 24 hours.
Materials Supplied (for serum samples)

Order No.

Volume

Component

ZV-4003-02R1-10

1 x 150 ml

Reagent 1, Contains internal standard

ZV-4003-02MP-10

2 x 2 L

Mobile Phase, Contains organic solvent

  ZV-4003-02WB-10

  1 x 0,5 L

Washing solution, Contains organic solvent

ZV-4003-KK-10

1 pc

User Guide

 
 
Materials Required But Not Supplied (for serum samples)

Order No.

Volume

Component

ZV-4003-02S1-10

1 x 3 ml

 Dried Blood Spot Calibrator Level 1

ZV-4003-02K1-10

1 x 3 ml

          Dried Blood Spot Control Level 1

ZV-4003-02K2-10

1 x 3 ml

          Dried Blood Spot Control Level 2

ZV-4003-02C1-10

1 pcs

Zivak PKU Serum HPLC Analytical Column

 
 
Materials Supplied (for dried blood samples)

Order No.

Volume

Component

ZV-4003-05R1-15

1 x 150 ml

Reagent 1, Contains internal standard

ZV-4003-05MP-15

2 x 2 L

Mobile Phase, Contains organic solvent

  ZV-4003-05WB-15

1 x 0,5 L

Washing solution, Contains organic solvent

ZV-4003-KK-Eng-15

1 pc

User Guide

 
 
Materials Required But Not Supplied (for dried blood samples)

Order No.

Volume

Component

ZV-4003-02S1-15

1 x 1 Spot

  Dried Blood Spot Calibrator Level 1

ZV-4003-02K1-15

1 x 1 Spot

     Dried Blood Spot Control Level 1

ZV-4003-02K2-15

1 x 1 Spot

     Dried Blood Spot Control Level 2

ZV-4003-02C1-15

       1 pcs

     Zivak PKU Dried Blood Spots HPLC Analytical Column

 
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User Manual 961.33 KB

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