IRMS ( Isotope Ratio Mass Spectrometer) Detection Protocol

IRMS equipment

The Biomedical Mass Spectrometry Facility is also equipped with a Finnegan Delta-S isotope ratio mass spectrometer (IRMS) which is providing breath CO₂ analyses, and hydrogen and oxygen analyses for the doubly labeled water method of determining energy expenditure.

Finnigan matt Delta S is connected to the HP5890 GC, which is used with the A200S Finnigan Matt automatic sampler, for IRMS analysis of CO₂. The software is ISODAT.

Standard Operation Procedure for CO₂, ¹⁸O, and D₂O from urine, plasma, and breath samples using the IRMS ( Isotope Ratio Mass Spectrometer)

1. Standard gas and isotopes:

H₂ standard- Z-Tech Trading Corporation ,9412 Rocky Branch Drive, Dallas, Texas, 75243.

O¹⁸- ISOTEC INC. A Matheson , USA company, water O¹⁸ normalized 10.3 atom %.

D2O- ISOTEC INC. 99.9% atom % D.

C¹³ urea- ISOTEC INC. 3858 Benner Rd, Miamisburg, OH, 45342.

2. GC conditions:

GC: COLOUM 每 HP Ultra 1 (Crosslinked Methyl Silicone Gum) 25m x 0.32 mm x 0.17 mm film thickness , USA patent #4,293,415, make in USA.

AUTO INJECTOR- Finigan Mat A200S.

Oven temp: Initial temp=100⁰C, initial time=0.00, rate=25⁰C /min, final temp=180⁰C, final time=0.20, injector temp=250⁰C.

3. Carrier gas and internal standard gas:

Compressed Helium ultra pure, compressed CO₂, and compressed air are from Air Liquide,2700 Post Oak Blvd, Houston, TX. 77056-8829.

4. Sample preparation procedure:

A. Breath CO₂ samples preparation (for ¹³C label.)

1.Collect patient＊s breath samples into the 1.8 ml EZ auto vial (WHEATON 1501 N 10^th St. Millville, NJ. 08332-2093 USA.) with a sip and stir straw ( Blue Diamond Straw, Inc. Los Angeles, Ca. 90065).

2. Crimp the vial with the aluminum seal, 11mm lined Tef/National Rubber (WHEATON 1501 N 10^thSt. Millville, NJ. 08332-2093 USA).

3.Samples are injected into the IRMS with the auto injector and the delta-13/12 is measured.

B. O¹⁸ samples preparation-

1. Pipet 200 ml of urine samples into an EZ auto vial, add 2ml of CO₂ gas using a 10cc syringe and crimp it tight quickly. Standard O¹⁸, tap H₂O and oral dose (1:500) are all treated the same as the samples.

1. All the samples are equilibrated for 48 hrs and let it shake on a shaker in room temperature with the speed of 200 (Vibrax, Serono Diagnostics.)

2. Samples are injected into the IRMS with the auto injector and the delta-18/16 is measured

C. Preparation of D₂O samples.

1. Put a 250ml flat bottom glass insert tube (HP, USA) into a 13X100 screw cap test tube.

2. Pipet 100ml of urine sample or plasma into the glass insert tube.

3. Cap the crew cap tube tight with a rubber liner cap, and incubate it in the 70⁰C heating block over night to distill the water in the samples.

4. Pipet 2ml of the distilled sample with a disposable micro-pipet and put it inside the prebaked 8§ long glass tube (Corning glass tubing), which contain about 0.5 gm of Zn.

5. The air inside the tubing is pumped down to about 30 millitorr with the vacuum system, and the tubing is cut and sealed with a torch.

6. The sealed tubing is then put into a furnace (Sybron/Thermolyne), and baked for 1 to 2 hours at 500⁰C.

7. The H₂ sample is introduced into the IRMS inlet directly by using the cracker, and the delta 2/3 is measured.