retardation in infants. A wide variety of analytical methods for the analysis of

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1 IN THE NAME OF GOD

2 Diagnosis and measurment of Phenylalanine in plasma and dried bilood spot (DBS) by a simple and sensitive HPLC method and compaire with Recipe Reference Health Laboratory Research Center, Ministry of Health and Medical Education, Tehran, IR Iran 2014.April..April.19 Nazeri SA., Khodaverdian k., Farzami M., Mohamadlo Tel Fax: nazeriali @yahoo.com M.

3 1-Introduction Diagnosis and measurement of phenylalanine in plasma and dried blood spot (DBS) is required for the diagnosis and subsequent dietary measurement of phenylketonuria (PKU) and it is important for treatment and prevention of mental retardation in infants. A wide variety of analytical methods for the analysis of amino acids have been developed over the years, however there is still a need for faster methods as well as for more sensitive multi-analyte methods. We developed d a simple and sensitive HPLC method for Diagnosis and Measurement phenylalanine in plasma and DBS.It is very cost effective and only standard HPLC equipment is needed.

4 A Simple and Sensitive Method for Measurement of Phenylalanine in Plasma & Dried Blood Spots (DBS) Laboratory Procedure Manual Analyte: Phenylalanine Matrix:DBS/Plasma Method: HPLC

5 2-Materials & Methods 2-2-Chemicals and reagents All materials and reagents (HPLCgrade) were purchased from Merck, Sigma and Recipe Co., Acetonitrile il and methanol were obtained from Merck Co.. Phenylalanine standard powder was obtained from Sigma Co. Recipe HPLC Kit (Germany), Recipe HPLC column, controls, calibrator, sample vial and filter paper were obtained from Recipe Co.(Germany). Blood Spot Controls were obtained from Kimia Pajouhan Co.

6 2-3-Methods -Mobile Phase and HPLC Conditions Agilent 1260 infinity series HPLC Pump: Isocratic, Flow rate: 1ml/min Column : C18 18, 15 Cm 4.6mm 5um Column Oven : 42 C Detector t : UV/VIS 214 nm Mobile Phase : Phosphate Buffer The mobile phase A for HPLC was prepared from 0.02 M sodium phosphate. While the coupling solution consisted of acetonitril (97:3,v/v),considering filtration before mixing. Post time was 4 min. The total run time was 12 min.

7 2-3-Methods 1- Phenylalanine Extraction (Recipe HPLC Kit) The frozen samples were allowed to thaw on ice for at least an hour. 50 µl of sample was added to Recipe sample vial that contains precipitation reagent and internal standard (IS), then incubated (15 min, 4 C),mixed on a vortex mixer for 10s, centrifuged (10000 rpm for 10min ). 20 µl of the supernatant fluid was injected into the HPLC system. The peaks were visible at 7.9 min for internal standard and 4.4 min for phenylalanine.

8 2-3-Methods 2-Phenylalanine extraction ( the HPLC method development) The frozen samples were allowed to thaw on ice for at least an hour. 50 µl of sample was added to a test tube, then 50 µl of internal standard (20 µmol/l), and 100 µl perchloric acid were added, mixed on a vortex for 30 s, centrifuged (10000 rpm for 6s ). 20 µl of the supernatant fluid was injected into the HPLC system. The peaks were visible at 7.5 min for Internal standard and 5.55 min for phenylalanine.

9 Measurement e e to of Phenylalanine yaa e from Filtre espot 1-Punch the filter sopt from middle of circle (5mm) 2- Put 50 µl Internal Standard ( 20 µmol/ L ) µl Precipitation Reagent & Vortex 10 sec. 3-Put the filter spot into the micro tube 4-Incubate (30 min, 37C) 5-Ultrasonic bath (10 min) 6-Centrifuge rpm,5 min 7-Inject 20 µl of supernatant into injector.

10 Measurement of Phenylalanine from Filter spot 5 mm punch

11 Dried Blood Spots Collection Problems

12 POTENTIAL ALIQUOT VARIABILITY WITH DRIED-BLOOD SPOT SPECIMENS 30 µl 30 µl = 5 mm punch 5 mm punch

13 Analysis Report Chromatogram

14 Analysis Report High Normal Low

15 3- Results 3-1- Validation of the HPLC method The values for control material (Recipe HPLC plasma controls were as follows: Observed (µmol/l) Target value (µmol/l) Range (µmol/l) LEVEL I LEVEL II Table 1: Values of Plasma control materials

16 3- Results 3-1- Validation of the HPLC method The values for control material (Blood Spot Controls - Kimia Pajouhan) were as follows: LEVEL I Observed (µmol/l) Target value (µmol/l) Range (µmol/l) LEVEL II LEVEL III Table 1: Values of Plasma control materials

17 3- Results 3-1- Validation of the HPLC method Precision Studies Obtained results for intra-assay & inter- assay studies were as follows in the tables below: ( 3.8%) The HPLC method development (µmol/l) (%CV) Level I ± Level II ± Level III ± Level IV ± intra-assay Table 1: Results for

18 3-Results - Validation of the HPLC method Precision Studies (5.4%) The HPLC method development elopment (µmol/l) (%CV) Level I ± Level II ± Level III ± Level IV ± Table 2 : Results for inter-assay

19 3-Results - Validation of the HPLC method Recovery Studies Stock standard solution (1000 ug/ml) in methanol was prepared and was kept in freezer (-20 C). Replicate extracts (n=3x) each of samples with and without "weighed in phenylalanine equivalent to 50,100 and 400 µmol/l were analyzed and the mean values used to calculate analytical recovery: %. (90-100%)

20 3-Results - Validation of the HPLC method Limit of Quantification Limit of Quantification (LOQ) of 0.2 mg/dl (13 µmol/l)for HPLC was obtained. This is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy and it is quoted as the concentration yielding a signal-to-noise ratio of 10:1. It is also is confirmed by analyzing a number of samples near this value (n=5).

21 3-Results - Validation of the HPLC method Linearity Studies For linearityit studies phenylalanine l concentrations ti of, 25, 50, 100, 200, 400, 800, 1600, 3200 were added. These samples were normal samples and analyzed with both systems. In the mentioned range the results for both system was linear mg/dl ( µmol/l)

22 Linearity Regression = 0.998

23 3-Results Methods Comparison For this purpose, thirty four DBS sample that received to Reference Health Laboratory Research Center for analysis by HPLC were selected. This patients t were suspected to phenylketonuria(pku)on new born screening program. Both system compared well and r 2 = 0.99 was obtained.

24 3-Results Methods Comparison 70 Series1 y = 1.002x R² = Linear (Series1)

25 4-Discussion The assay is linear from 0.2 mg/dl to 50 mg/dl with an intraasay CV lower than 6% and Interassay CV Lower than 5%. LOQ = 0.2 mg/dl. Method comparison showed that correlation between Recipe HPLC Kit and development method in RHL was very good ( r = 0.999).

26 4-Discussion The method has a wide linear range, good precision, and provides results that correlate well with Recipe HPLC Kit, the sample volume is low, sample preparation is simple, rapid and analysis is fast, also the method is very cost effective.therefore the procedure has provide reliable in regular routine use.

27 Thanks for your attention