arnitine / Acylcarnitines L-MS/MS Analysis Kit (Dried lood ... · • Switch off the pump and...

34
D. No: ZV-3051-KK-15_Rev01 Carnitine / Acylcarnitines LC-MS/MS Analysis Kit (Dried Blood Spot) ZV-3051-0200-15 200 2-8 °C User Manual

Transcript of arnitine / Acylcarnitines L-MS/MS Analysis Kit (Dried lood ... · • Switch off the pump and...

Page 1: arnitine / Acylcarnitines L-MS/MS Analysis Kit (Dried lood ... · • Switch off the pump and connect the column in flow direction. • Activate the method and allow mobile phase(s)

D. No: ZV-3051-KK-15_Rev01

Carnitine / Acylcarnitines LC-MS/MS Analysis Kit (Dried Blood Spot)

ZV-3051-0200-15

200

2-8 °C

User Manual

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Table Of Contents

Page 1. INTENDED USE: ...................................................................................................................................................... 3

2. SUMMARY AND EXPLANATION: ..................................................................................................................... 3

3. TEST PRINCIPLE: ................................................................................................................................................... 3

4. WARNING AND PRECAUTIONS: ...................................................................................................................... 4

5. STORAGE AND STABILITY: ................................................................................................................................ 4

6. MATERIALS SUPPLIED: ....................................................................................................................................... 4

7. MATERIALS REQUIRED BUT NOT SUPPLIED: ........................................................................................... 5

8. PROCEDURE NOTES: ............................................................................................................................................ 6

9. LIMITATIONS OF THE PROCEDURE: ............................................................................................................. 7

10. PRE-TEST SET-UP INSTRUCTIONS: ............................................................................................................... 8

11. TEST PROCEDURE: ................................................................................................................................................ 9

12. QUALITY CONTROL: .......................................................................................................................................... 10

13. CALCULATION OF RESULTS:.......................................................................................................................... 10

14. INTERPRETATION OF RESULTS: ................................................................................................................. 11

15. EXPECTED VALUES: ........................................................................................................................................... 11

16. LC-MS/MS PARAMETERS: ............................................................................................................................... 12

17. MS SCAN PARAMETERS: .................................................................................................................................. 13

18. ANALYTICAL PERFORMANCE: ...................................................................................................................... 15

19. SAMPLE CHROMATOGRAM: ........................................................................................................................... 16

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1. INTENDED USE:

Carnitine / Acylcarnitines LC-MS/MS analysis kit for dried blood spot.

2. SUMMARY AND EXPLANATION:

Carnitine / Acylcarnitines screening is the most important part of the clinical diagnosis of amino acid, fatty acid or organic acid metabolism. Carnitine / Acylcarnitines screening allows to the diagnosis of over 30 metabolic diseases from a drop of blood samples taken with a screening card. This screening test occurred by measurement of Carnitine / Acylcarnitins with isotope dilution tandem mass spectrometry. LC-MS/MS is a powerful tool for gives results Carnitine / Acylcarnitines in 1.5 minutes. Screening of following analytes can be performed by Zivak Carnitine / Acylcarnitine LC-MS/MS Analysis Kit and Zivak NeoZin Screening software. Main methods and procedures that have been selected are based on EN ISO 14971. By the quantitative analysis of carnitine esters including such as free carnitine, C0, C2, C4, C6, C8, C10, C12, C14, C16, C16 OH, C18 , C18 OH carnitine disorders which are related with fatty acid oxidation.

By the quantitative analysis of carnitine esters including such as C3, C3DC, C4OH, C5, C5OH,C5DC carnitine, disorders which are related with organic acid metabolism.

3. TEST PRINCIPLE:

Carnitine / Acylcarnitines are extracted from dried blood spot with an organic solvent contains internal standards. Then extracted analytes derivatised via reagents. Derivatisated Carnitine / Acylcarnitines are analysed by Tandem Mass spectrometry.

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4. WARNING AND PRECAUTIONS:

• For in-vitro diagnostic use only. For professional use only. • Before you start, read the instructions carefully. • In case of damage of the kit package, please contact Zivak or your supplier. • Do not use expired kits and components. Please check batch no and expiry date before you start. • Protective gloves and goggles should be worn. • When working with biological fluids, please take any necessary precautions to prevent infection with blood borne pathogens. Appropriate bio-safety precautions and disposal of bio hazardous wastes should be followed • Please check the labels on reagent bottles. Reagents of this kit contains hazardous material may cause eye and skin irritations. • All calibrators and controls of this kit which contain human blood or dried blood was tested and found negative for HIV 1/2 and HCV antibodies, HbsAg, HIV 1/2 and HCV genome. Nevertheless, the blood controls should be considered as potentially infectious and treated with appropriate care. • It’s recommended to work in a fume hood for your safety as Reagent 2 is volatile and please avoid inhaling.

5. STORAGE AND STABILITY:

• This analysis kit can be shipped at room temperature. • Controls , Reagent 2, 3 should be stored at 2-8 °C. • Reagent 1 should be stored at -20 °C. • All other components of the kit 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.

6. MATERIALS SUPPLIED:

Order No. Volume Symbol Component

ZV-3051-02R1-15 1 x 40 ml

Reagent 1, Contains deuterated internal standards

R1

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ZV-3051-02R2-15 1 x 12 ml

Reagent 2, Contains derivatisation reagent

ZV-3051-02R3-15 1 x 40 ml

Reagent 3, Contains acid

ZV-3051-02WB-15 1 x 0,5 L

Washing solution, Contains organic solvent

ZV-3051-02MP-15 1 x 0,6 L

Mobile Phase, Contains organic solvent

ZV-3051-KK-15 1 x 1 pc

User Manual

7. MATERIALS REQUIRED BUT NOT SUPPLIED:

ZV-3051-02K1-15 1 x 1 Spot

Dried Blood Spot Control Level 1, Contains Human Blood

ZV-3051-02K2-15 1 x 1 Spot

Dried Blood Spot Control Level 2, Contains Human Blood

ZV-3051-02S1-15 1 x 1 Spot

Dried Blood Spot Calibrator Level 1, Contains Human Blood

ZV-3051-02S2-15 1 x 1 Spot

Dried Blood Spot Calibrator Level 2, Contains Human Blood

• Zivak Carnitine / Acylcarnitines Dried Blood Spots LC-MS/MS Trap Column

Housing • Carnitine / Acylcarnitines Dried Blood Spots LC-MS/MS Trap Column Cartridge • 20-200 µL pipette

R3

MP

R2

WB

KK

Control

Level 1

ControlLevel 2

II

Calibrator Level 1

Calibrator

Level 2

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• Pipette tips • 3.5 mm puncher • Conical autosampler vial or vial insert • User manual

8. PROCEDURE NOTES: Any improper handling of samples or modification of the test procedure may influence the results. The indicated pipetting volumes, incubation times, temperatures and pre-treatment steps have to be performed strictly according to the instructions. Use calibrated pipettes and devices only. Once the test is started, all steps should be completed without interruption. Make sure that required reagents, materials and devices are prepared ready at the appropriate time. Allow all reagents and specimens to reach room temperature (18-25 °C) and gently swirl each vial of liquid reagent and sample before use. Mix reagents without foaming. Avoid contamination of reagents, pipettes and wells/tubes. Use new disposable plastic pipette tips for each reagent, standard or specimen. Do not interchange caps. Always cap not used vials. Do not reuse wells/tubes or reagents. Incubation time affects results. All tubes or wells should be handled in the same order and time sequences.

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9. LIMITATIONS OF THE PROCEDURE:

Specimen collection has a significant effect on the test results. Please always follow the

universal safety precautions for accurate and safe DBS sampling given below.

Universal Safety Precautions for DBS sampling:

• Treat all blood samples as though they are infectious

• Wash hands, wear gloves and apron/lab coat

• Take precaution to avoid needle injury

• Dispose of contaminated sharps and waste appropriately

• Clearly label each card with appropriate identification number. It is

unacceptable to submit a blood card for testing that has not been properly labelled.

• Apply gentle pressure to the heel and allow a large drop of free flowing blood to

collect at the puncture site.

• Working quickly, hold the filter paper by the edges and touch the filter paper

gently against the large drop of blood and in one step allow a sufficient quantity of

blood to soak through and completely fill or saturate a circle.

• A completed saturated spot will contain approx.100 µl of blood.

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• Repeat, until you have collected enough blood to fill at least 3 circles on the

blood collection card.

• It is critical that entire circle be uniformly saturated with blood.

• If collecting spots using a pipette, collect 100 µl of blood and gently apply to

filter paper.

Due to it is a screening method any result with an elevated concentration has to be

indicated as ‘presumptive positive’ and has to be confirmed with further sampling and

confirmatory testing.

A false negative result of this assay cannot be excluded with absolute certainty.

10. PRE-TEST SET-UP INSTRUCTIONS:

Set-up the Instrument: • Purge the HPLC pumps with a high flow rate of mobile phase(s). This should be done by pumping the mobile phase(s) through the system for 2 minutes at a flow rate of 4.0 ml/min. • Switch off the pump and connect the column in flow direction. • Activate the method and allow mobile phase(s) to flow through the pre-filter for 5 minutes. • Make sure the bottle of mobile phase bottle is closed well, otherwise components of the mobile phase could evaporate; this alters the retention times.

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11. TEST PROCEDURE:

Sample Pre-treatment (Manual)

Take dried blood spot sample in 3.5 mm diameter by a puncher on a 96-well microplate.

Add 200 L of R1 to each well. Incubate for 30 minutes at room temperature.

After incubation, take the liquid phase on another well. Then dry with warm air flow.

Add 60 L of R2 on dried samples.

Cover microplate properly with alumina foil or appropriate seal And incubate for 20 minutes at 65 °C.

Remove alumina foil and dry the liquid phase completely with warm air flow.

Add 200 µl of R3 to dissolve the dried wells.

Take the liquid into a vial or directly inject 20 µl from 96 well plate to LC-MS/MS system.

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

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12. QUALITY CONTROL:

The test results are only valid if the test is performed by the following the instructions.

Moreover, the user must strictly adhere to the rules of GLP (Good Laboratory Practice)

or other applicable standards/laws. Kit controls have to be found within the acceptable

ranges as stated on the QC (Quality Control) Certificate. If the criteria are not met, the

run is not valid and should be repeated. Each laboratory should use known samples as

further controls.

In case of any deviation, the following technical issues should be proven: Expiration

dates of (prepared) reagents, storage conditions, pipettes, devices, incubation

conditions and washing methods. It is recommended to participate at appropriate

quality assessment trials.

13. CALCULATION OF RESULTS:

Isotop dilution method is used for calculation of results. The analyte concentrations calculated against area of their internal standard with known concentrations. Using Zivak NeoZin Data Evaluation Software is very easy and effective software to analyse complex screening data.

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14. INTERPRETATION OF RESULTS:

Various societies for Carnitine / Acylcarnitines screening recommend different Cut-Off

values for repetition of the measurement and the application of confirmatory assays.

Depending on the application of samples of different populations of newborns it is

highly recommended that each laboratory establishes its own range of normal values

and that this distribution of values is co-ordinated with the recommendations of the

responsible society of this geographic region.

The results themselves should not be the only reason for any therapeutic

consequences. They have to be correlated to other clinical observations and diagnostic

tests.

15. EXPECTED VALUES:

Expected values are set in the Zivak NeoZin Data Evaluation Software. It is recommended that each laboratory establishes its own range of normal values.

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16. LC-MS/MS PARAMETERS:

Device Zivak Tandem Gold LC-MS/MS System

Column Zivak Carnitine / Acylcarnitines Screening Dried Blood Spots LC-MS/MS Trap Column Housing - Cartridge

Injection Volume 20 µL

Pump Program 00:00 min 100% B 01:30 min 100% B

Flow 0.30 mL/min

Ionization Mode: ESI positive

CID Gas 2.0 mTorr

API Nebulizing Gas 55 psi

Scan Time 1.519 sec

SIM Width 1.5 amu

Drying Gas 30 psi

Drying Gas Temp. 350 °C

Needle 5000V

Shield 600V

Capillary 30V

Detector 1500 V

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17. MS SCAN PARAMETERS:

Zivak Carnitine / Acylcarnitines Screening Dried Blood LC-MS/MS Analysis kit contains above analytes in controls.

No Analyte MH+ (m/z) MS/MS Capillary (eV) CE (eV) 1 C0 Carnitine 218.30 85.00 30 20 2 C0 Carnitine-D9 227.30 85.00 30 20 3 C2 Carnitine 260.30 85.00 30 20 4 C2 Carnitine-D3 263.30 85.00 30 20 5 C3 Carnitine 274.30 85.00 30 20 6 C3DC Carnitine 360.30 85.00 30 20 7 C3 Carnitine-D3 277.30 85.00 30 20 8 C4 Carnitine 288.30 85.00 30 20 9 C4OH Carnitine 304.30 85.00 30 20

10 C4 Carnitine-D3 291.30 85.00 30 20 11 C5 Carnitine 302.30 85.00 30 20 12 C5DC Carnitine 388.30 85.00 30 20 13 C5OH Carnitine 318.30 85.00 30 20 14 C5 Carnitine-D9 311.30 85.00 30 20 15 C6 Carnitine 316.50 85.00 30 20 16 C8 Carnitine 344.30 85.00 30 20 17 C8 Carnitine-D3 347.30 85.00 30 20 18 C10 Carnitine 372.30 85.00 30 20 19 C12 Carnitine 400.30 85.00 30 20 20 C14 Carnitine 428.30 85.00 30 20 21 C14 Carnitine-D9 437.50 85.00 30 20 22 C16 Carnitine 456.40 85.00 30 20 23 C16OH Carnitine 472.40 85.00 30 20 24 C16 Carnitine-D3 459.50 85.00 30 20 25 C18 Carnitine 484.50 85.00 30 20 26 C18OH Carnitine 500.50 85.00 30 20

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INTERNAL STANDARD CONCENTRATIONS

Analyte Final Concentration

(µmol/L)

C0 Carnitine-D9 1.52

C2 Carnitine-D3 0.38

C3 Carnitine-D3 0.076

C4 Carnitine-D3 0.076

C5 Carnitine-D9 0.076

C8 Carnitine-D3 0.076

C14 Carnitine-D9 0.076

C16 Carnitine-D3 0.152

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18. ANALYTICAL PERFORMANCE:

Analytical Specificity (Cross Reactivity): No cross-reactivities were found with the typical substances tested.

No Analyte LOD

(µmol/L) LOQ

(µmol/L) Accuracy

(%) Intra-Assay

Precision (%CV)

Inter-Assay Precision

(%CV)

Linearity (R2)

1 C0 Carnitine 0.02 0.07 96.7 4.02 5.2 0.985

2 C2 Carnitine 0.04 0.13 93.3 3.21 4.1 0.996

3 C3 Carnitine 0.02 0.07 98.3 2.32 3.3 0.994

4 C4 Carnitine 0.05 0.16 98.6 4.46 5.1 0.993

5 C5 Carnitine 0.02 0.07 97.6 3.36 4.3 0.994

6 C4OH Carnitine 0.05 0.14 95.6 3.22 4.1 0.996

7 C6 Carnitine 0.04 0.12 95.5 2.46 3.2 0.994

8 C5OH Carnitine 0.02 0.07 97.2 3.52 4.2 0.991

9 C8 Carnitine 0.09 0.26 96.9 2.46 3.5 0.992

10 C3DC Carnitine 0.07 0.20 92.7 1.27 2.5 0.996

11 C10 Carnitine 0.09 0.26 92.7 1.39 2.7 0.993

12 C5DC Carnitine 0.08 0.23 96.8 1.54 2.6 0.997

13 C12 Carnitine 0.07 0.20 93.5 2.26 3.5 0.996

14 C16OH Carnitine 0.09 0.13 95.7 3.42 4.5 0.996

15 C14 Carnitine 0.07 0.20 95.4 2.14 3.7 0.994

16 C16 Carnitine 0.08 0.23 94.5 3.57 4.6 0.993

17 C18OH Carnitine 0.09 0.08 93.1 3.27 3.9 0.993

18 C18 Carnitine 0.04 0.13 96.4 3.45 4.2 0.993

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19. SAMPLE CHROMATOGRAM:

for Carnitine / Acylcarnitines

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Sample chromatograms are taken from Carnitine / Acylcarnitines Screening Neozin Data Evaluation Software.

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ACYL CARNITINES: C0-Carnitine:

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Karnitin / Açil Karnitinler LC-MS/MS Analiz Kiti (Kuru Kanda)

ZV-3051-0200-15

200

2-8 °C

Kullanma Kılavuzu

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İÇERİK Sayfa

1. KULLANIM AMACI: ............................................................................................................................................. 21

2. ÖZET VE AÇIKLAMA: ......................................................................................................................................... 21

3. TEST PRENSİBİ .................................................................................................................................................... 22

4. UYARILAR VE ÖNLEMLER .............................................................................................................................. 22

5. SAKLAMA KOŞULLARI VE KARARLILIK .................................................................................................... 22

6. KİT İÇERİĞİ ............................................................................................................................................................ 23

7. KİT İÇERİĞİ DIŞINDA GEREKLİ OLAN MALZEMELER ......................................................................... 24

8. ÇALIŞMA PROSEDÜRÜ ...................................................................................................................................... 24

9. YÖNTEMDEKİ KISITLAMALAR...................................................................................................................... 25

10. ENSTRÜMAN ÖN AYARLARI. ......................................................................................................................... 25

11. NUMUNE HAZIRLIĞI .......................................................................................................................................... 26

12. KALİTE KONTROL .............................................................................................................................................. 26

13. SONUÇLARIN HESAPLANMASI ..................................................................................................................... 27

14. SONUÇLARIN YORUMLANMASI.................................................................................................................... 27

15. BEKLENEN DEĞERLER ..................................................................................................................................... 27

16. MS/MS PARAMETRELERİ ............................................................................................................................... 28

17. MS SCAN PARAMETRELERİ............................................................................................................................ 29

18. ÖRNEK KROMATOGRAM ................................................................................................................................. 31

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1. KULLANIM AMACI:

Kuru kan örneklerinde Karnitine / Açil Karnitinler tarama için LC-MS/MS analiz kiti.

2. ÖZET VE AÇIKLAMA:

Karnitine / Açil Karnitinler tarama, aminoasit, yağ asitleri ve organik asit metabolizmalarındaki bozuklukların klinik tanısında en büyük rolü oynar. Karnitine / Açil Karnitinler tarama ile 30’dan fazla metabolik hastalığın teşhisini yapmak mümkündür. Bunun için bir damla kan örneği yeterlidir. Bu test bazı Karnitine / Açil Karnitinlerin izotop seyreltme yöntemine dayanarak tandem kütle spektrometrisi ile yapılır. LC-MS/MS sistemi Karnitine / Açil Karnitinler tarama için Zivak Karnitine / Açil Karnitinler Tarama Analiz kiti ile bütün bu analitlerin sonucunu 1.5 dakika içinde verebilen çok güçlü bir sistemdir.

Aşağıdaki analitlerin tamamı Zivak Karnitine / Açil Karnitinler LC-MS/MS Analiz Kiti ve Zivak NeoZin software ile yapılabilir.

Yağ asidi oksidasyonunda rol oynayan serbest karnitinler ve karnitin esterleri: C0, C2, C4, C6, C8, C10, C12, C14, C16, C16 OH, C18 , C18 OH karnitin.

Organik asit metabolizmasını rol oynayan karnitin esterler: C3, C3DC, C4OH, C5, C5OH,C5DC karnitin..

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3. TEST PRENSİBİ

Karnitine / Açil Karnitinler organik çözücü içeren internal standart aracılığı ile kuru

kandan ekstrakte edilir. Ekstrakte edilen analitler reaktifler ile türevlendirilir.

Türevlendirilmiş Karnitine / Açil Karnitinler LC-MS/MS sisteminde analiz edilir.

4. UYARILAR VE ÖNLEMLER

Sadece in-vitro kullanım amaçlıdır. Profesyonel kullanım içindir. Çalışmaya başlamadan önce, kullanım kılavuzunu dikkatlice okuyunuz. Kit ambalajının zarar görmüş olması durumunda, lütfen Zivak veya bayinizle irtibata geçiniz. Son kullanma tarihi geçmiş kitleri veya bileşenlerini kullanmayınız. Kullanmadan önce seri numarasını ve son kullanma tarihini kontrol ediniz. Toksik bileşen konsantrasyonları çok düşük olmakla birlikte güvenlik amacıyla çeker ocak altında çalışılması önerilir. Çalışırken koruyucu eldiven ve gözlük kullanılmalıdır. Çalışılan kan veya biyolojik örneklerden kaynaklanabilecek enfeksiyonlara karşı gerekli her türlü önlemi alınız. Biyo-zararlı atıklar için uygun biyo-güvenlik ve atık yönetimi koşullarını sağlayınız. Lütfen kullanmadan önce reaktif şişeleri üzerindeki etiketleri kontrol ediniz. Bu kitin reaktifleri göze ve cilde zarar verecek maddeler içerir.

5. SAKLAMA KOŞULLARI VE KARARLILIK

R1 (-20) °C’de saklanmalıdır. R2, R3 ve kontroller 2-8 °C ‘de muhafaza edilebilir. Kitin

diğer bileşenleri oda sıcaklığında saklanabilir. Bütün bileşenler önerilen koşullarda

saklandığı takdirde son kullanma tarihine kadar garantilidir..

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6. KİT İÇERİĞİ

Sipariş No Hacim Sembol İçerik

ZV-3051-02R1-15 1 x 40 ml

Reaktif 1,

ZV-3051-02R2-15 1 x 12 ml

Reaktif 2,

ZV-3051-02R3-15 1 x 40 ml

Reaktif 3,

ZV-3051-02WB-15 1 x 0,5 L

Yıkama Solüsyonu

ZV-3051-02MP-15 1 x 0,6 L

Mobil Faz

ZV-3051-KK-15 1 x 1 ad

Kullanma Klavuzu

R3

MP

R1

R2

WB

KK

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7. KİT İÇERİĞİ DIŞINDA GEREKLİ OLAN MALZEMELER

ZV-3051-02K1-15 1 x 1 Spot

Kurukan Spotu Kontrol Level 1,

ZV-3051-02K2-15 1 x 1 Spot

Kurukan Spotu Kontrol Level 2,

ZV-3051-02S1-15 1 x 1 Spot

Kurukan Spotu Kalibrator Level 1,

ZV-3051-02S2-15 1 x 1 Spot

Kurukan Spotu Kalibrator Level 2,

KİT İÇERİĞİ DIŞINDA GEREKLİ OLAN MALZEMELER Zivak Karnitine / Açil Karnitinler Guard Kolon ve kartuş 20-200 µL pipet Pipet uçları 3,5mm puncher Konik autosampler viali veya vial insert Kullanım Kılavuzu

8. ÇALIŞMA PROSEDÜRÜ

Test prosedürünün yanlış ve değiştirilerek uygulanması test sonucunu etkileyebilir.

Pipetleme hacimleri, inkübasyon zamanları ve hazırlık aşamaları tam olarak

Calibrator Level 1

Calibrator

Level 2

Control

Level 1

ControlLevel 2

II

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belirtilen miktarlarda ve talimatlara uygun bir biçimde yapılmalıdır. Her zaman ve

sadece kalibre edilmiş pipetler ve cihazlar kullanılmalıdır.

Teste bir kere başlandığı zaman, herhangi bir kesintiye uğramadan

tamamlanmalıdır. Bunu sağlamak için gerekli reaktiflerin, maddelerin ve cihazların

zamanında hazır olduğundan emin olunuz. Çalışmaya başlamadan önce bütün

reaktiflerin ve numunlerin oda sıcaklığına (18-25 °C) gelmesini bekleyiniz, sıvı

reaktifleri ve örneklerini kullanmadan önce hafifçe çalkalayarak karıştırınız.

Reaktifler, pipetler ve numune hazırlık tüplerinde kirlilik ve bulaşma olmamasına

dikkat ediniz. Her reaktif, standart ve numune için ayrı ve yeni pipet ucu kullanınız.

Vial kapaklarını kendi aralarında değiştirmeyiniz. Kullanılmadığı zaman vial

kapaklarını her zaman kapalı tutunuz. Vialler, vial kapakları, numune hazırlık

tüpleri ve pipet uçları tek kullanımlıktır.

İnkübasyon süreleri sonuçları etkilemektedir. Bütün numune hazırlık işlemleri

belirtilen sırada ve sürede yapılmalıdır.

9. YÖNTEMDEKİ KISITLAMALAR

Konsantrasyonalarda görülen herhangi bir artış tespit edildiğinde ‘şüpheli pozitif ‘ olarak değerlendirilmeli ve tekrar numune alınıp test tekrar edilmelidir. Bu çalışmada herhangi yalancı negatif bir sonuç kesinlikle göz ardı edilemez. Numune toplama ve saklama koşulları test sonuçlarını belirgin bir şekilde etkilemektedir. Daha sonraki günlerde çalışılacak tam kan numuneleri analiz yapılana kadar dondurulmuş olarak saklanmalıdır.

10. ENSTRÜMAN ÖN AYARLARI.

HPLC pompalarına mobil faz bağlandıktan sonra 4ml/dk akış ile 2 dakika

boyunca purge yapılmalıdırlar. Metodu aktif ettikten sonar sistemin şartlanması için 15 dk beklenmelidir.

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11. NUMUNE HAZIRLIĞI

1.Kurutulmuş kan örneğinden 3,5 mm çapında örnek alınarak 96-well microplate üzerinde bir kuyucuğa konulur.

3. İnkübasyondan sonra sıvı kısım başka bir kuyucuğa alınır. Sıcak hava akımı ile kurutulur. 4. f 2 kurutulmuş numunelerin üzerine eklenir. Mikroplate’in üzeri alüminyum folyo ile iyice kapatılır. 20 dakika 65 °C’ de bekletilir. 5. Alüminyum folyo çıkarılır. Kuyucuktaki sıvı sıcak hava akımı ile tamamen kurutulur.

erek kurutulmuş kısım çözülür. 20 µl LC-MS/MS sistemine enjeksiyon yapılır.

12. KALİTE KONTROL

Test sonuçları ancak verilen test talimatlarına uyulduğu takdirde geçerlidir.

Kullanıcılar çalışırken GLP veya diğer kurumlar tarafından belirlenmiş olan

kurallara ve standartlara bağlı kalmalıdır.

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Bütün standart ve kontrol değerleri kalite kontrol sertifikasında belirtilen aralıklarda

olmalıdır. Değerler bu aralıkların dışında kaldığı takdirde analiz geçerli değildir ve

tekrarlanmalıdır.

Her laboratuvar daha ileri aşama kontroller için değeri bilinen numuneleri

kullanmalıdır.

Bu değerlerden sapma olması halinde reaktiflerin son kullanma tarihi, saklama

koşulları, kullanılan pipetlerin doğruluğu, cihazlar, inkübasyon koşulları ve yıkama

metotları gibi teknik detayların doğruluğundan emin olunmalıdır.

Ayrıca uygun dış kalite kontrol programına üye olunması önerilir.

13. SONUÇLARIN HESAPLANMASI

Sonuçların hesaplanmasında izotop seyreltme metodu kullanılır. Analitlerin

konsantrasyonları, konsantrasyonu bilinen internal standart alanlarına göre

hesaplanır. Zivak NeoZin Newborn Screening Data Evaluation Software karmaşık

tarama datalarını çok kolay ve etkili bir şekilde hesaplayan bir yazılımdır.

14. SONUÇLARIN YORUMLANMASI

Farklı toplumlarda farklı Cut-Off değerleri belirlenmelidir. Farklı toplumlardaki hasta

numuneleri için yapılan çalışmalara bağlı olarak, her laboratuvarın kendi normal

değerlerini belirlemesi önemle tavsiye edilir ve bu değerlerin dağılımı o coğrafi

bölgedeki sorumlu kurumun önerdiği değerler ile uyumlu olmalıdır.

Tanı ve tedavi aşamalarında bu analizlerin sonuçları klinik gözlemler ve tanı

testleri ile desteklenmelidir.

15. BEKLENEN DEĞERLER

Her laboratuvarın kendi normal değerlerini belirlemesi önemle tavsiye

edilir.

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.

16. MS/MS PARAMETRELERİ

Cihaz Zivak Tandem Gold LC-MS/MS Sistem

Kolon Zivak- Karnitine / Açil Karnitinler Kolon

Enjeksiyon Hacmi 20 µL

Gradiyant 00.00 dk 100% B 0,30 ml/dk 01:30 dk 100% B 0,30 ml/ dk

Akış 0,30 mL/dk

Iyonizasyon Modu ESI positive

CID Gazı 2,0 mTorr

API Nebulizing Gaz 55 psi

Tarama Süresi 1,519 sn

SIM Width 1,5 amu

Drying Gaz 30 psi

Drying Gaz.Sıc 350 °C

İğne Voltajı 5000V

Shield Voltajı 600V

Capillary 30V

Dedektör 1500

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17. MS SCAN PARAMETRELERİ

Zivak Kurukanda Karnitine / Açil Karnitinler LC-MS/MS Analiz Kiti kontrolleri yukarıda listelenen analitleri içermektedir.

No Analit MH+ (m/z) MS/MS Capillary (eV) CE (eV) 1 C0 Carnitine 218.30 85.00 30 20 2 C0 Carnitine-D9 227.30 85.00 30 20 3 C2 Carnitine 260.30 85.00 30 20 4 C2 Carnitine-D3 263.30 85.00 30 20 5 C3 Carnitine 274.30 85.00 30 20 6 C3DC Carnitine 360.30 85.00 30 20 7 C3 Carnitine-D3 277.30 85.00 30 20 8 C4 Carnitine 288.30 85.00 30 20 9 C4OH Carnitine 304.30 85.00 30 20

10 C4 Carnitine-D3 291.30 85.00 30 20 11 C5 Carnitine 302.30 85.00 30 20 12 C5DC Carnitine 388.30 85.00 30 20 13 C5OH Carnitine 318.30 85.00 30 20 14 C5 Carnitine-D9 311.30 85.00 30 20 15 C6 Carnitine 316.50 85.00 30 20 16 C8 Carnitine 344.30 85.00 30 20 17 C8 Carnitine-D3 347.30 85.00 30 20 18 C10 Carnitine 372.30 85.00 30 20 19 C12 Carnitine 400.30 85.00 30 20 20 C14 Carnitine 428.30 85.00 30 20 21 C14 Carnitine-D9 437.50 85.00 30 20 22 C16 Carnitine 456.40 85.00 30 20 23 C16OH Carnitine 472.40 85.00 30 20 24 C16 Carnitine-D3 459.50 85.00 30 20 25 C18 Carnitine 484.50 85.00 30 20 26 C18OH Carnitine 500.50 85.00 30 20

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INTERNAL STANDART KONSANTRASYONLARI

Final Konsantrasyonlar (µmol/L)

C0 Carnitine-D9 1,52

C2 Carnitine-D3 0,38

C3 Carnitine-D3 0,076

C4 Carnitine-D3 0,076

C5 Carnitine-D9 0,076

C8 Carnitine-D3 0,076

C14 Carnitine-D9 0,076

C16 Carnitine-D3 0,152

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18. ÖRNEK KROMATOGRAM

Karnitinler için

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C0-Carnitine: