arnitine / Acylcarnitines Dried lood Spots L-MS/MS...

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D. No: ZV-3051-KK-15_Rev08

Carnitine / Acylcarnitines Dried Blood Spots

LC-MS/MS Analysis Kit User Manual

ZV-3051-0200-15

200

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D. No: ZV-3051-KK-15_Rev08

Table of Contents 1. INTENDED USE ......................................................................................................... 3

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

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

4. WARNING AND PRECAUTIONS ............................................................................ 3

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 ................................................................... 6

10. PRE-TEST SET-UP INSTRUCTIONS .................................................................... 7

11. TEST PROCEDURE .................................................................................................. 7

12. QUALITY CONTROL ................................................................................................. 8

13. CALCULATION OF RESULTS ................................................................................. 8

14. INTERPRETATION OF RESULTS .......................................................................... 8

15. EXPECTED VALUES................................................................................................. 9

16. LC-MS/MS PARAMETERS ....................................................................................... 9

17. MS SCAN PARAMETERS ...................................................................................... 10

18. ANALYTICAL PERFORMANCE ............................................................................ 11

19. SAMPLE CHROMATOGRAM ................................................................................ 12

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D. No: ZV-3051-KK-15_Rev08

1. INTENDED USE

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

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.

4. WARNING AND PRECAUTIONS

• For in-vitro diagnostic use only.

• For professional use only.

• Read the instructions carefully, before you start.

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D. No: ZV-3051-KK-15_Rev08

• In case of damage of the kit package, please contact Zivak or your supplier.

• Do not use expired kits and components.

• Please check the batch no and expiry date before start.

• Protective gloves and goggles should be worn.

• Please take any necessary precautions to prevent infection with blood borne

pathogens while working with biological fluids. Appropriate bio-safety precautions and

disposal of bio-hazardous wastes should be followed.

• Please check the labels on reagent bottles. Reagents of this kit contain 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

• It’s recommended to work in a fume hood for your safety as Reagent 2 is volatile and

please avoid inhaling.

• 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

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

Reagent 2, Contains derivatization reagent

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

Reagent 3, Contains acid

R3

R1

R2

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D. No: ZV-3051-KK-15_Rev08

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

Materials listed in the table below are required and should be ordered separately.

Order No. Volume Symbol Component

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

Dried Blood Spot Control Level 1

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

Dried Blood Spot Control Level 2

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

Dried Blood Spot Calibrator Level 1

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

Dried Blood Spot Calibrator Level 2

ZV-3051-02C1-15 1 x 1 pcs Zivak Carnitine / Acylcarnitines Dried Blood Spots LC-MS/MS Trap Column Holder

ZV-3051-CSPE-15 1 x 1 pcs Carnitine / Acylcarnitines Dried Blood Spots LC-MS/MS Trap Column Cartridge

• 20-200 µL pipette • Pipette tips • 3.5 mm puncher • Conical autosampler vial or vial insert

MP

WB

KK

Control

Level 2

Calibrator

Level 1

Calibrator

Level 2

Control

Level 1

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D. No: ZV-3051-KK-15_Rev08

8. PROCEDURE NOTES

Any inappropriate 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 has been started, all steps should be completed without interruption. Make sure that

required reagents, materials and devices are prepared ready at the appropriate time.

Leave aside 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 the vial caps. Always keep vials

closed when not been used. Do not re-use wells/tubes or reagents.

Incubation time affects results. All tubes or wells should be handled in the same order and time

sequences.

9. LIMITATIONS OF THE PROCEDURE

Specimen collection and storage have 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. • 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.

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D. No: ZV-3051-KK-15_Rev08

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.

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.

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D. No: ZV-3051-KK-15_Rev08

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.

12. QUALITY CONTROL

The test results are only valid if the test has been performed by following the instructions. Moreover

the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable

standards/laws. All standards and kit controls must 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:

Expiry 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.

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.

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D. No: ZV-3051-KK-15_Rev08

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.

16. LC-MS/MS PARAMETERS

Device LC-MS/MS Analysis 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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D. No: ZV-3051-KK-15_Rev08

17. MS SCAN PARAMETERS

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

INTERNAL STANDARD CONCENTRATIONS

Analyte Final Concentration

(µmol/L)

C0 Carnitine-D9 1.52








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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D. No: ZV-3051-KK-15_Rev08

18. ANALYTICAL PERFORMANCE

Analytical Specificity (Cross Reactivity): No cross-reactivity was 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 0,023 0,070 98,7 4,02 0,14 0,9983

2 C2 Carnitine 0,042 0,126 99,7 3,21 0,04 0,9984

3 C3 Carnitine 0,024 0,073 99,7 2,30 0,01 0,9994

4 C4 Carnitine 0,054 0,163 98,8 4,44 0,12 0,9993

5 C5 Carnitine 0,023 0,070 99,0 3,44 0,00 0,9937

6 C4OH Carnitine 0,047 0,142 95,0 3,12 0,08 0,9997

7 C6 Carnitine 0,041 0,122 100,6 2,50 0,14 0,9978

8 C5OH Carnitine 0,022 0,066 102,4 3,40 0,15 0,9957

9 C8 Carnitine 0,088 0,263 99,7 3,41 0,06 0,9943

10 C3DC Carnitine 0,068 0,205 104,6 3,66 0,23 0,9989

11 C10 Carnitine 0,088 0,265 105,3 4,95 0,21 0,9962

12 C5DC Carnitine 0,076 0,227 101,7 2,87 0,15 0,9969

13 C12 Carnitine 0,068 0,205 104,7 4,08 0,52 0,9984

14 C16OH Carnitine 0,090 0,269 96,7 4,45 0,14 0,9967

15 C14 Carnitine 0,067 0,200 95,3 3,78 0,08 0,9945

16 C16 Carnitine 0,077 0,232 100,2 1,39 0,02 0,9943

17 C18OH Carnitine 0,091 0,273 95,0 3,84 0,19 0,9936

18 C18 Carnitine 0,042 0,127 100,4 3,67 0,37 0,9948

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D. No: ZV-3051-KK-15_Rev08

19. SAMPLE CHROMATOGRAM

for Carnitine / Acylcarnitines

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D. No: ZV-3051-KK-15_Rev08

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

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D. No: ZV-3051-KK-15_Rev08

ACYL CARNITINES: C0-Carnitine:

arnitine / Acylcarnitines Dried lood Spots L-MS/MS...

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