PROPERTIES AT A GLANCE

Zivak LC-MS/MS Phenylketonuria analysis kit is designed and produced for the determination of phenylalanine, tyrosine and tryptophan levels in dried blood spots with a high specificity and analytical sensitivity within a single run.

• This method is powerful tool which can be used both as a screening test and also for the quantitative determination of phenylalanine, tyrosine and tryptophan in dried blood spots with high specificity and sensitivity. 
• The phenylalanine, tyrosine and tryptophan determinations with this method have a high analytical sensitivity (LOD, 0.18, 0.21 and 0.20 nmol/mL, respectively; LOQ, 0.54, 0.63, and 0.60 nmol/mL).
• The amino acid determinations have a very high specificity of 100 %.
• The method is very suitable for clinical laboratory settings owing to a single step sample preparation and a 2 minute run time. 12 samples within one hour and 200 samples within a day (8 hours) can be analyzed.
• Sample preparation does not require deproteinization.   
• Zivak LC-MS/MS Phenylketonuria analysis kit is designed and produced to be used with Tandem Gold Mass Spectrometer.
• The kit contains enough reagents and materials to analyze 200 samples.
• Zivak LC-MS/MS Phenylketonuria analysis kit can be used in the screening, diagnosis and follow-up of phenylketonuria, hyperphenylalaninemia, tyrosinemia and other nutritional or metabolic diseases which alter the blood levels of these amino acids. 
• Zivak LC-MS/MS Phenylketonuria analysis kit is produced in accordance with the TS ISO 9001:2000 and TS ISO 13485:2003 quality standards.
  
  

INTENDED USE

The essential amino acid phenylalanine, which is supplied through the diet is used up during protein synthesis or converted to tyrosine by the enzyme phenylalanine hydroxylase. The deficiency of this enzyme, or its cofactors, or the presence of mutant forms with low activity result in phenylketonuria which is characterized by the accumulation of phenylalanine and decrease of tyrosine in the body. The accumulated phenylalanine is converted to more soluble forms such as phenylketones and phenylamines which are excreted with urine. If the presence of the disease which is inherited autosomal recessively can not be diagnosed early, the accumulation of phenylalanine and phenylketones, and the inadequate supply of tyrosine during growth ususally result in irreversible motor and mental development retardation. Since the unwanted effects of this disease can be prevented with dietary modifications, it is very important to diagnose the affected children as early as possible after birth. Therfore the screening of all newborn babies for PKU is necessary for the sake of the baby, the family and the community.

SAMPLE AND REAGENT PREPARATION PROCEDURE

Preparation of the working reagent:

R1 and R2 is mixed in appropriate volumes according to the number of samples to be analyzed as in table below:

Number of samples	R2 (µL)	R1 (µL)
2	360	40
5	900	100
10	1800	200
20	3600	400

Preparation of the calibrators:

Add 3 mL of deionised water to the calibrator vial. Vortex until its completely dissolved.

1. Pipet 20 mL of dissolved plasma calibratör to an eppendorf  tube.

2.  Add 800 mL of working reagent (R1 + R2) and vortex for 10 seconds.

3. Incubate at +4 °C for 15 minutes.

4. Centrifuge at 10000 x g for 5 minutes.  

5. Pipet 500 µL of the supernatant into a sample preparation vial. Inject 5 µL into the Tandem Gold LC-MS/MS.

Preparation of the samples:

This method is designed to be used with dried blood spots on paper. The samples must be kept at 4 °C until analyzed.

1. A sample 4 mm in diameter should be punched from the dried blood paper and put into an eppendorf tube.

2. Add 200 µL of working reagent (R1 + R2) and vortex for 10 seconds.

3. Incubate for 30 minutes in a water bath at 37 °C.

4. Vortex for 10 seconds.

5. Centrifuge at 10000 x g for 5 minutes.

6. Pipet 150 µL of supernatant into the small sample adaptor in sample preparation vials. Inject 5 µL into the Tandem Gold LC-MS/MS.

MEASUREMENT PARAMETERS

Amino Acid	Symbol	MW	RT	LOD (nmol/mL)
Phenylalanine	PHE	165	0,66	0,18
Phenylalanine -D5	PHE-D5	170	0,64
Tyrosine	TYR	181	0,41	0,21
Tyrosine-D4	TYR-D4	185	0,41
Tryptophan	TRP	204	1,08	0,20
Tryptophan -D5	TRP-D5	209	1,03

CHROMATOGRAMS


PKU LC-MS/MS ANALYSIS KIT

Dried blood spots (200 tests)

Catalog No

Contents

Reagents

CCatalog No	Product	Volume	Quantity
Z ZV-3003-02S1-15	Plasma Calibrator  (S1)	3 mL	1 / kit
ZV-3003-02K1-15	Dried Blood Control Level I (C 1)	1 x 6 spot	1 / kit
ZV-3003-02K2-15	Dried Blood Control LevelII (C 2)	1 x 6 spot	1 / kit
ZV-3003-02R1-15	Reagent 1 (R1, internal standard)	5 mL	1 / kit
ZV-3003-02R2-15	Reagent 2 (R2)	50 mL	1 / kit
ZV-3003-02MP-15	Mobile Phase A	1 L	1 / kit

Materials

CCatalog No	Product	Volume	Quantity
ZV-3003-02C1-15	Zivak-PKU Column		1 / kit
ZV-9022-0004	4 mmpunch		4 / kit
ZV-9011-0250	1,5 mL eppendorf tube	250 /pack	1 / kit
ZV-9001-0100	Sample preparation vial(1,8 mL)	100/ pack	2 / kit
ZV-9002-0100	sample preparation vial cap  (1,8 mL)	100/ pack	2 / kit
ZV-9003-1000	20-200 µL  pipet tip	1000/ pack	2 / kit
ZV-9004-0250	100-1000 µL  pipet tip	250/ pack	1 / kit
ZV-9014-0001	20-200 µL  pipet		1 / kit
ZV-9015-0001	100-1000 µL pipet		1 / kit

Materials not supplied

Centrifuge for eppendorf tubes and  vortex mixer.

Storage and Stability

C1, C2 and S1 should be kept at 4°C until dissolved and at -20 °C after dissolving. R1 should be kept at 4 °C. Other reagents should be kept at room temperature. When kept as suggested in the user manual, all reagents will be stable until the expiry date.

Warnings and precautions

As a general precaution all sample preparation steps should be carried out in a safety hood, wearing protective gloves and glasses. When working with biological samples, necessary steps should be taken to avoid contamination with bloodborne pathogens. All samples posing a biological threat should be appropriately disposed of.  

RESULTS

The results can be expressed in nmol/mL or mmol/dL, as deseired.

LIMITATIONS OF THE PROCEDURE

PERFORMANCE CHARACTERISTICS

Analytical Sensitivity

 

Amino Acid	LOD (nmol/mL)	LOQ (nmol/mL)
Phenylalanine	0,18	0,54
Tyrosine	0,21	0,63
Tryptophan	0,20	0,60

LOD:Limit of Detection

LOQ:Limit of Quantitation

Specificity

The specificty of the method for these amino acids is 100 %.

Precision

CV for PHE is:  % 2,3

CV for TYR is:  % 2,1

CV for TRP is:  % 1,9

Linearity

The correlation coefficient (R²) for linearity of PHE determinations between 0-100 nmol/mL is 0,998.

The correlation coefficient (R²) for linearity of TYR determinations between 0-100 nmol/mL is 0,999.

The correlation coefficient (R²) for linearity of TRP determinations between 0-100 nmol/mL is 0,998.

Recovery

The recovery rate for PHE is 95 %.

The recovery rate for TYR is 96 %.

The recovery rate for TRP is 94 %.

Interference

The presence of hemoglobin, lipids and bilirubin does not interfere with the method.

Zivak LC-MS/MS Phenylketonuria analysis kit is designed and produced to be used with Tandem Gold Mass Spectrometer.