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Quantitative Determination of Phenylketonuria

Quantitative Determination of Phenylketonuria
CE-IVD certified and suitable for any kind of LC-MS/MS instrument

 

The Zivak Phenylketonuria (PKU) LC-MS/MS analysis kit can be used in combination with the Zivak-Multitasker as well as manually. Analysis run time for the manual version is 2 minutes. Due to the efficient and quite smart procedure of the Zivak-Multitasker, the automated version guarantees to receive quantitative results in just 2,5 minutes per test, including sample preparation-, sample injection- and analysis run time. Additionally, high accuracy is being rectified through external quality tests.

 

LC-MS/MS analysis kit for the in-vitro diagnostic quantitative determination of phenylketonuria in human serum & dried blood samples. 

Phenylketonuria (PKU) is an autosomal recessive genetic disorder characterized by a deficiency in the enzyme phenylalanine hydroxylase (PAH). This enzyme is necessary to metabolize the aminoacid phenylalanine to the amino acid tyrosine. When PAH is deficient, phenylalanine accumulates on the brain tissue and is converted into phenylpyruvate (also known as phenylketone), 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.

Zivak PKU LC-MS/MS kit was developed for quantitative detection of Phenylketonuria in human serum, plasma and dried blood samples. LC-MS/MS is a effective tool for the study of metabolic disorders (e.g. PKU). LC-MS/MS analysis gives results in 2 minutes with minimal sample preparation. Main methods and procedures that is selected by Zivak are based on EN ISO 14971 .

Quantitative Determination of Phenylketonuria 0
LC-MS/MS Parameters (for serum samples)

Device

Zivak Tandem Gold LC-MS/MS System

Column

Zivak PKU Serum LC-MS/MS Analytical Column 

Injection Volume

5 µL

Pump Program

0:00    min                   100 % B

2:00    min                   100 % B

Flow

0.30 mL/min

Ionization Mode

ESI positive

CID Gas

2.4 mTorr

API Nebulizing Gas

55 psi

Scan Time

0.342 sec

SIM Width

1.2 amu

Drying Gas

35 psi

Drying Gas Temp.

350 °C

Needle

5000V

Shield

600V

Capillary

50V

Detector

1700 V

 
 
LC-MS/MS Parameters (for dried blood samples)

Device

Zivak Tandem Gold LC-MS/MS System

Column

Zivak PKU Dried Blood Spots LC-MS/MS Analytical Column 

Injection Volume

5 µL

Pump Program

0:00    min                   100 % B

2:00    min                   100 % B

Flow

0.30 mL/min

Ionization Mode

ESI positive

CID Gas

2.4 mTorr

API Nebulizing Gas

55 psi

Scan Time

0.342 sec

SIM Width

1.2 amu

Drying Gas

35 psi

Drying Gas Temp.

350 °C

Needle

5000V

Shield

600V

Capillary

50V

Detector

1700 V

 
 
Analytical Performance

No

Analyte

LOD (µmol/L)

LOQ (µmol/L)

Accuracy (%)

Intra-Assay

Precision

 (%CV)

Inter-Assay

Precision

(%CV)

Linearity

(R2)

1

Phenylalanine (PHE)

0.18

0.54

95

2.3

2.6

0.998

2

Tryptophan (TRP)

0.20

0.60

94

2.1

2.5

0.996

3

Tyrosine (TYR)

0.21

0.63

96

1.9

2.3

0.997

 

Storage & Stability:

  • This analysis kit can be shipped at room temperature.
  • After arrival, calibrator and controls, Reagent 1, 2, 3, and 4 can be stored at 2-8 °C.
  • MP, WB can be stored at room temperature.
  • Reagent 2, 3 and 4 are volatile, please make sure that the lids of reagents are closed tightly after use.
  • 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 serum calibrator/control and dissolve.

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

 

Sample Pretreatment (Serum):

Take 50 mL of serum calibrator into a 2 ml capped preparation.

 

Add 100 mL of R1. Mix by vortex for 10 seconds.

 

Add 200 mL of R2. Mix by vortex for 10 seconds.

 

Add 25 µl of R3. Mix by vortex for 10 seconds. Wait for 1 minute. Mix by vortex for 10 seconds.

 

Add 800 µl of R4. Mix by vortex for 10 seconds.

 

Centrifuge at 8.000 x rpm for 5 minutes.

 

Take 500 µl upper liquid into autosampler vial.

 

Inject 5 mL to the LC-MS/MS system.

 

 

Note: The prepared sample is stable at 2-4 °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 capped preparation tube.

 

Add 200 µL of R1. Mix by vortex for 10 seconds. Mix by vortex for 5 sec. Incubate for 30 minutes at room temperature.

 

Add 200 mL of R2. Mix by vortex for 10 seconds.

 

Add 25 µl of R3. Mix by vortex for 10 seconds. Wait for 2 minute. Mix by vortex for 10 seconds.

 

Add 800 µl of R4. Mix by vortex for 10 seconds.

 

Centrifuge at 12.000 x rpm for 2 minutes.

 

Take 500 µl upper liquid into autosampler vial.

 

Inject 5 mL to the LC-MS/MS system.

Note: The prepared sample is stable at 2-4 °C for 24 hours.
Materials Supplied (for serum samples)

Order No.

Volume

Component

ZV-3003-02R1-10

1 x 20 ml

Reagent 1, Contains deuterated internal standards

ZV-3003-02R2-10

1 x 40 ml

Reagent 2, Contains acid

ZV-3003-02R3-10

1 x 5 ml

Reagent 3, Contains derivatisation reagent

ZV-3003-02R4-10

4 x 40 ml

Reagent 4, Contains dilution reagent

ZV-3003-02MP-10

1 x 0,5 L

Mobile Phase, Contains organic solvent

ZV-3003-02WB-10

  1 x 0,5 L

Washing solution, Contains organic solvent

ZV-3003-KK 10

1 x 1 pc

User Guide

 
 
Materials Required But Not Supplied (for serum samples)

Order No.

Volume

Component

ZV-3003-02S1-10

1 x 3 ml

Lyophilized Serum Calibrator Level 1

ZV-3003-02K1-10

1 x 3 ml

Lyophilized Serum Control Level 1

ZV-3003-02K2-10

1 x 3 ml

Lyophilized Serum Control Level 2

ZV-3003-02C1-10

1 pcs

Zivak PKU Serum LC-MS/MS Analytical Column

 
 
Materials Supplied (for dried blood samples)

Order No.

Volume

Component

ZV-3003-02R1-15

1 x 20 ml

Reagent 1, Contains deuterated internal standards

ZV-3003-02R2-15

1 x 40 ml

Reagent 2, Contains acid

ZV-3003-02R3-15

1 x 5 ml

Reagent 3 Contains derivatisation reagent

ZV-3003-02R4-15

4 x 40 ml

Reagent 4, Contains dilution reagent

ZV-3003-02MP-15

1 x 0,5 L

Mobile Phase, Contains organic solvent

ZV-3003-02WB-15

1 x 0,5 L

Washing solution, Contains organic solvent

ZV-3003-KK 10

1 x 1 pc

User Guide

,
 
Materials Required But Not Supplied (for dried blood samples)

Order No.

Volume

Component

ZV-3003-02S1-15

1 x 1 spot

Dried Blood Spot Calibrator Level 1

ZV-3003-02K1-15

1 x 1 spot

Dried Blood Spot Control Level 1

ZV-3003-02K2-15

  1 x 1 spot

Dried Blood Spot Control Level 2

ZV-3003-02C1-15

  1 pcs

Zivak PKU Dried Blood Spots LC-MS/MS Analytical Column

 
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User Manual 972.76 KB

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