500 μM succinylacetone (μL)
Water (μL)
Succinylacetone (nmol)
Cal 1
0
1500
0
Cal 2
25
1475
12.5
Cal 3
50
1450
25
Cal 4
75
1425
37.5
Cal 5
100
1400
50
Cal 6
200
1300
100
2.4 Internal Standards
1.
2.5 mM 3,4,5,6,7-13C5-succinylacetone (13C5-SA) internal standard stock solution: Dissolve 10 mg 13C5-SA in 24.4 mL deionized water. Store at −80 °C indefinitely.
2.
250 μM 3,4,5,6,7-13C5-succinylacetone (13C5-SA) internal standard working solution: Dilute 5 mL of 2.5 mM 13C5-SA stock solution with 45 mL of deionized water. Aliquot into 15 mL tubes, store at −80 °C. Keep one aliquot at 4 °C, protected from light for up to 3 months.
2.5 Quality Controls
1.
Number and frequency: 2 levels of quality controls (QC) are run with every analytical batch.
2.
Preparation and storage of QC materials
Level 1 or QCL: 25 mL commercially purchased drug free human urine sample pool.
Then succinylacetone concentration of 100 mmol/mol creatinine is equal to N/1130. Calculation is as follows:
(a)
Measure the blank urine creatinine level in mg/mL as N mg/mL or N/113 mol/L.
(b)
Then succinylacetone concentration of 100 mmol/mol creatinine is equal to N/1130.
Add (N/1130) × 158.15 × 25 mg succinylacetone to 25 mL blank urine. Calculate sample volume (containing 1 μmol creatinine) for one assay, make small aliquots with a bit more than you need for one assay and store at −80 °C until exhausted.
3.
Validation and certification of QC lot: Target values for new control lot are established from 20 assays performed with the current controls. The acceptable range is defined as mean ± 2 SD.
4.
Maintaining QC Lot: QC tracking lot must be created and maintained by person routinely performing test. When inventory is low (<30 aliquots), new QC lot needs to be prepared and validated prior to clinical use.
2.6 Supplies
1.
15 mL (16 × 125 mm) screw-cap glass tubes.
2.
10 mL (16 × 100 mm) screw-cap glass tubes.
3.
Transfer pipettes.
4.
Auto sampler vials with caps (VWR 9301-0978 and 5182-0550).
5.
Nitrogen gas.
6.
Helium gas.
7.
GC column: Agilent HP-5 column with dimensions of 25 m × 0.20 mm × 0.33 μm.
2.7 Equipment
1.
Agilent 7890 Gas Chromatography/5975 Mass Spectrometry (GC/MS) system with autosampler.
2.
Glas-Col Evaporator.
3.
Single-tube Vortex.
4.
Multi-tube Vortex.
5.
Eppendorf 5702 centrifuge.
3 Methods
3.1 Stepwise Procedure
1.
Remove patient samples and quality control samples from freezer to thaw at room temperature. Set the temperature of the evaporator to 70 °C.
2.
Prepare the worksheet: enter the creatinine values of the samples and calculate the sample volumes as equivalent to 1 μmol creatinine (0.113/creatinine in mg/mL).
3.
Label one set of 15 mL glass tubes and two sets of 10 mL glass tubes (blank, QCL, QCH, patient samples) in numerical order as in the worksheet.
4.
Pipette samples into labeled 15 mL tubes as per the volume calculated in the worksheet.
5.
Add water to each sample as per calculations in the worksheet to bring the final volume up to 1.5 mL.
6.
Add 100 μL 250 μM 13C5-SA internal standard (25 nmol) to all samples.
7.
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Start oximation reaction by adding:
(a)
