ETHIL ACETATE in urine by GC-FID
Code GC01510
BIOCHEMISTRY
The ethyl acetate at room temperature is presented as a volatile liquid, colorless and with
a pleasant fruity smell, a compound is highly flammable and irritating. Is widely used as a
solvent for paints, resins and glues and it is relatively less toxic than other chlorinated
solvents. The proposed method determines the ethyl acetate inject in GC / FID a rate of
urine sample previously treated in an appropriate solvent.
EUREKA srl – LAB DIVISION
VAT N° 01547310423
E-mail:[email protected]
www.eurekaone.com
Head Quarter:
Via Enrico Fermi 25
60033 Chiaravalle (AN) ITALY
Tel. +39 071 7450790
Fax + 39 071 7496579
This product fulfills all the requirements of Directive 98/79/EC on in vitro diagnostic medical devices (IVD). The declaration of
conformity is available upon request.
Release N° 001
Ethyl Acetate in urine by GC/FID
1
March 2011
TECHNICAL FEATURES
Principle of the Method :
This method allows to determine the urinary ethyl acetate by GC-FID. The ethyl acetate is first diluted and then
is directly injected into GC. aliquot of urine sample previously treated in an appropriate solvent.
Recovery:
>95%
Sensitivity:
30 mg/l
Dynamic Range of the Method:
30 – 15.000 mg/l
Reference Values:
n.d.
Components of the kit :
All Reagents are ready to use and stable 3 years at ≤
20°C.
Reagent A – Stabilization Solution, 1 x 150 ml
Reagent B – Test Solution / Chemical Standard, 1 x 5 ml
Store at 2-8 °C
Minimum Instrumental equipment required:
GC/FID
Operational Computer
Optional Equipment:
Autosampler
Urine Collection Procedure:
After work shift, collect 10 ml of urine in a suitable test
tube.
Stable 4 weeks at –20 °C.
2
IMPORTANT: USE ONLY IN GLASS TUBE EVERY STEP - DO NOT USE PLASTIC TUBES
PREANALYTICAL PROCEDURE
Preparation of Test Solution.
Dispense in a 10ml glass tube:
•
•
450 µl of Reagent A – Stabilization Solution
50 µl of Reagent B – Test Solution
Vortex for 10 sec.
The samples are injected directly transferring the supernatant into vials for GC
Inject 2 µl of solution into gas chromatograph GC. Verify that the Test solution has retention time similar to
fig. 1. If the Test is all right you can start with the analytical procedure; if not, check the functionality of the
analytical system.
ANALYTICAL PROCEDURE
STEP 1: Preparation of Calibration Standard at 450 mg/l
Dispense in a glass tube of 10ml:
•
•
200 µl of Water
200 µl of Reagent B – Test Solution
Mix at Vortex
Dispense in a glass tube:
•
•
900 µl of Urine without Ethyl Acetate
100 µl of Test Solution previously diluted
IMPORTANT: This Calibration Standard Solution is prepared from time to time
To verify that the urine is effectively free from Ethyl Acetate, to prepare a Blank:
•
•
900 µl of Urine
100 µl of H2O HPLC grade
If the Blank urine presented the peak of Ethyl Acetate, proceed to identify the quantitative difference
between the areas / heights of the peaks of Ethyl Acetate in urine enriched and blank.
Release N° 001
Ethyl Acetate in urine by GC/FID
3
March 2011
Dispense in a glass tube:
Calibration
Standard
100 µl
Calibration Standard
Control
Sample
Blenck
Reagent A – Stabilization Solution
Control
Sample
Blanck
100 µl
100 µl
900 µl
900 µl
900 µl
100 µl
900 µl
Close vials hermetically
•
Centrifuge 2 minutes at 5000 rpm
•
Put all the surnatant in vials for GC-FID. Inject 2 µl in GC-FID.
Prepare work list and start the serie according to the system’s specifications.
Release N° 001
Ethyl Acetate in urine by GC/FID
4
March 2011
ETHYL ACETATE - Warnings
REAGENT B : TEST SOLUTION / CHEMICAL STANDARD
ETHYL ACETATE
9000 mg/l
SET OF GAS-CROMATOGRAPH:
•
•
•
•
•
•
•
Factor Four VF- 624 ms Column 30 m x 0,32 mm x 1,80 µm (conditioned)
Injector Temperature 280 °C
Splitting Ratio:
In: OFF
0’ ON:10
Temperature 40 °C x 3,9 minutes + 20 °C/min till up 150 °C x 0 minutes + 50
°C/min till up 280 °C x 0 minutes (run 12 minutes)
Column Flow: 1 ml/min
Detector Temperature (FID): 240 °C
Range (sensitivity) Detector: 12 (max)
CONDITIONING OF FACTOR FOUR VF-624 MS COLUMN:
Follow the manufacturer prescription.
CLEANING OF THE COLUMN ANF SYRINGE:
Before injection wash the syringe with ACETONE.
Disconnect the detector. Keep the column at the highest temperature for the
recommended time. (See the manufacturer ’s instructions)
OPERATIONAL COMPUTER PARAMETERS:
IN CONFORMITY WITH THE SPECIFICATION OF OPERATIONAL COMPUTER SOFTWARE
5
ETHYL ACETATE IN URINE
(Reference Chromatograms)
µV
145.000
10.000
9.800 µV
9.600
9.400
9.200
9.000
8.800
8.600
8.400
8.200
8.000
7.800
7.600
7.400
7.200
7.000
6.800
6.600
6.400
6.200
6.000
5.800
5.600
5.400
5.200
5.000
4.800
4.600
4.400
4.200
4.000
3.800
3.600
3.400
3.200
3.000
2.800
2.600
2.400
2.200
2.000
1.800
1.600
1.400
1.200
1.000
800
600
400
200
0
-200
08 T Acet ETILE 9000mgL new prep1.DATA
140.000
135.000
E
LI
T
E
i
d
O
T
A
T
E
C
A
130.000
125.000
120.000
115.000
110.000
105.000
100.000
95.000
90.000
85.000
80.000
75.000
70.000
65.000
60.000
55.000
50.000
45.000
40.000
35.000
30.000
25.000
20.000
15.000
10.000
5.000
0
-5.000
RT [min]
0
0,5
1
1,5
2
Fig. 1 :
2,5
3
3,5
4
4,5
5
5,5
6
6,5
7
7,5
8
8,5
9
9,5
10
10,5
11
11,5
2
12
Test Solution
R.T. 4.9 ETHYL ACETATE
28 Ur Cert Acet ETILE 450mgL new prep1.DATA
E
LI
T
E
ot
at
e
c
A
RT [min]
2,2
2,4
2,6
2,8
Fig. 2 :
7
3
3,2
3,4
3,6
3,8
4
4,2
4,4
4,6
4,8
5
5,2
5,4
5,6
5,8
6
Calibration Standard
R.T. 4.9
ETHYL ACETATE
6,2
6,4
6,6
6,8
7
7,2
450 mg/l
7,4
7,6
7,8
8
ETHYL ACETATE IN URINE
(Reference Chromatograms)
10.000
9.800 µV
9.600
9.400
9.200
9.000
8.800
8.600
8.400
8.200
8.000
7.800
7.600
7.400
7.200
7.000
6.800
6.600
6.400
6.200
6.000
5.800
5.600
5.400
5.200
5.000
4.800
4.600
4.400
4.200
4.000
3.800
3.600
3.400
3.200
3.000
2.800
2.600
2.400
2.200
2.000
1.800
1.600
1.400
1.200
1.000
800
600
400
200
0
-200
2
27 Ur Cert Acet ETILE 225mgL new prep1.DATA
E
LI
T
E
ot
at
e
c
A
RT [min]
2,2
2,4
2,6
Fig. 3 :
2,8
3
3,2
3,4
3,6
3,8
4
4,2
4,4
4,6
4,8
5
5,2
5,4
5,6
Urine Sample
R.T. 4.9 ETHYL ACETATE
8
5,8
6
6,2
6,4
6,6
225 mg/l
6,8
7
7,2
7,4
7,6
7,8
8