TETRACHLOROMETHANE IN URINE BY GC/MS Headspace
Code GC17510
BIOCHEMISTRY
The Tetrachloromethane (CCl4) (or Carbonium Tetrachlorinated), also known as Freon 10, is a
synthetic compound used in laboratories as a solvent, in agriculture as a pesticide and chemical
industries as an intermediate in the production of fluorocarbons and cleaning of metals. In the past
it was used for dry cleaning and degreasing as. At room temperature is a nonpolar liquid is volatile
and highly toxic because it is able to trigger radical chain reactions that degrade cell membranes.
The tetrachloromethane is absorbed by inhalation, ingestion and dermal, as it is eliminated through
the breath and the urine. Exposure to high concentrations of tetrachloromethane even in vapor
form, affects the central nervous system, cause headaches and in severe cases can lead to coma
and death. The proposed method determines the tetrachloromethane injected into GC-MS /
headspace aliquot of urine sample previously treated in an appropriate solvent. The levels of
tetrachloromethane are determined on a urine sample at the beginning and end of shift.
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° 003
Tetrachloromethane in urine by GC/MS-headspace
1
July 2012
TECHNICAL FEATURES
Principle of the Method :
This method determines the tetrachloromethane urinary GC / MS headspace. The tetrachloromethane is first
diluted and then is directly injected into GC.
Recovery :
100%
Sensitivity :
3,4 µg/l by MS
Dynamic Range of the Method:
3,4 – 10.000 µg/l by MS
Normal Values:
n.d.
CV%:
2,6% intra serie
5,8% inter serie
Components of the kit :
All the reagents are ready-to-use and stable 3 years
at 2 - 8°C.
Reagent A – Diluting Solution, 1 x 100 ml
Reagent B – Test Solution / Chemical Standard, 2 x 5 ml
For the sampling of Reagent C, the cap
sealing requires the use of 100 ul Hamilton
syringe Cod. Z81000
Store at 2-8 °C
Minimum Instrumental equipment required:
GC/MS
Operational Computer
Optional Equipment:
Autosampler
Urine Collection Procedure:
After work shift, collect 10 ml of urine in a suitable
test tube.
Stable 1 day at 4 °C.
Stable 2 weeks at –20 °C.
2
PREANALYTICAL PROCEDURE
Preparation of Test Solution
Dispense in a vial of 10ml for headspace:
•
•
•
1000 µl of Reagent A – Diluting Solution
800 µl of Water HPLC grade
200 µl of Reagent B – Test Solution (using for 2 times Hamilton syringe of 100 µl)
Inject 1000 µl of solution into gas chromatograph GC. Verify that the Test solution has retention time similar
to fig. below. 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 Solution at 125 µg/l
Dispense in a vial of 10ml for headspace:
•
•
•
975 µl of Urine without Tetrachloromethane
25 µl of Reagent B – Chemical Standard (using Hamilton syringe of 100 µl)
1000 µl of Reagent A – Diluting Solution
Quickly close with a special airtight cap and vortex for 5 sec
Wash the Hamilton syringe with Methanol after use
IMPORTANT: This Calibration Standard Solution is prepared from time to time
To verify that the urine is effectively free from Tetrachloromethane, to prepare a Blank:
•
•
1000 µl of Urine
1000 µl of Reagent A – Diluting Solution
Quickly close with a special airtight cap and vortex for 5 sec
If the Blank urine presented the peak of Tetrachloromethane, proceed to identify the quantitative
difference between the areas / heights of the peaks of Tetrachloromethane in urine enriched and
blank.
•
1000 µl of the samples are directly injected.
Release N° 003
Tetrachloromethane in urine by GC/MS-headspace
3
July 2012
Preparation of unknown samples
Dispense in a vial of 10ml for headspace:
•
•
1000 µl of Urine
1000 µl of Reagent A – Diluting Solution
Quickly close with a special airtight cap and vortex for 5 sec
Inject in headspace 1 ml of Calibrator, the possible levels of control and unknown samples previously
prepared and start the series according to the specifications of the system.
N.B.: at the end of the preparation of standard solution immediately close the Test / Chemical
Standard with parafilm and stored immediately at 2 - 8 ° C.
Release N° 003
Tetrachloromethane in urine by GC/MS-headspace
4
July 2012
TETRACHLOROMETHANE - Warnings
REAGENT B : TEST SOLUTION / CHEMICAL STANDARD
TETRACHLOROMETHANE
5.000 µg/l
SET OF AUTOSAMPLER :
•
•
•
•
•
•
•
•
•
•
•
•
Syringe Temperature: 85 °C
Agitator Temperature: 85 °C (incubate for 3 minutes at 750 rpm)
Agitator ON: 5 sec
Agitator OFF: 2 sec
Plunger Fill Speed: 500 µl/sec
Fill Strokes: 0
Viscosity Delay: 1 sec
Pre-Injection Delay: 0 sec
Plunger Injection Speed: 1000 µl/sec
Post-Injection Delay: 0.1 sec
Syringe Flush Time: 60 sec
GC Cycle Time (for prep ahead): 19 min
SET OF GAS-CROMATOGRAPH:
•
•
•
•
•
Restek Rxi-5SILms Column 30 m x 0,25 mm, 0,25 µm (conditioned)
Injector’s Temperature 230 °C
Temperature 60 °C x 2 minutes + 8 °C/min up to 100 °C x 0 min + 40 °C/min up to 280
°C x 0 min (run 12 min)
Helium Gas 1 ml/min
Split Report Start: Initial ON split 10
0.0 min OFF
0.03 min ON split 30
3 min ON split 10
SET OF MASSA DETECTOR (4000):
•
•
•
•
•
•
Range of Massa 30 – 140
Operative Method in Electronic Impact (EI)
Temperature of Massa: 190 °C
Temperature of Manifold: 80 °C
Temperature of Transfer Line: 270 °C
Filament on: 0.2 minute
CONDITIONING OF RESTEK COLUMN
Follow the manufacturer prescription.
Do not condition column/s if connected to the Massa Detector.
CLEANING OF THE COLUMN
At the end of every analytical session bring the gradient at the maximum temperature of the
column (recommended by the manufacturer) with a speed of 30 ° C / minute Storage: at Room
T° with septa in both ends in order to avoid the entry of air into the column.
OPERATIONAL COMPUTER PARAMETERS
IN CONFORMITY WITH THE SPECIFICATION OF OPERATIONAL COMPUTER SOFTWARE
N.B.: We recommend using a syringe of the autosampler for headspace ONLY for the injections of
tetrachloromethane to avoid any carryover effects and / or interfering due to other types of methods.
5
TETRACHLOROMETHANE IN URINE
(Reference Chromatograms/Spectrums GC-MS / Headspace)
kCounts
Spectrum1A
BP: 117.1 (228660=100%), test 1.000_6-27-2012.sms
CarbonTetrachloride 7u125 1_6-22-2012.sms Ions: 117.0 Filtered
30:140
150
1.689 min, Scan: 386, 30:140, Ion: 1065us, RIC: 758825, BC
117.1
228660
100%
125
75%
1A
100
75
50%
121.0
73124
25
4 4 8 . 6 R F 1 . 6 8 9 m in
C a r b o n T e t r a c h lo r id e
50
32.6
21972
0
84.1
36132
47.4
30417
0%
2.5
Fig. 1 :
82.1
57607
25%
5.0
7.5
Calibration Standard
R.T. 1.7 TETRACHLOROMETHANE
10.0
25
minutes
Fig. 2 :
125 µg/l
7
50
75
100
Massa Spectrum of Calibration Standard
MOLECULAR IONS : 117-121-82
125
m/z
TETRACHLOROMETHANE IN URINE
(Reference Chromatograms/Spectrums GC-MS / Headspace)
Spectrum1A
BP: 117.1(228660=100%), test 1.000_6-27-2012.sms
Carbon Tetrachloride1 u31.25 1_6-22-2012.sms Ions: 117.0 Filtered
kCounts
30:140
150
1.689 min, Scan: 386, 30:140, Ion: 1065us, RIC: 758825, BC
117.1
228660
100%
125
75%
100
1A
75
50%
121.0
73124
50
82.1
57607
25
49 3 .1 R F 1. 68 4 m i n
C a r b o n T e t ra c h l o ri d e
25%
32.6
21972
0
0%
2.5
Fig. 3 :
84.1
36132
47.4
30417
5.0
7.5
Urine Sample
R.T. 1.7 TETRACHLOROMETHANE
10.0
25
minutes
Fig. 4 :
25 µg/l
8
50
75
100
Massa Spectrum of Urine Sample
MOLECULAR IONS : 117-121-82
125
m/z