Quantification of Dihydroxyacetone Phosphate (DHAP) in Human Red Blood Cells by HPLC-TripleTOF 5600™ Mass Spectrometer

 

2°(μl)

3° (μl)

RBC matrix

Final conc. (μM)

Cal 1
 
100

q.s. to 10 ml

 10

Cal 2

100
 
q.s. to 10 ml

100

Cal 3

200
 
q.s. to 10 ml

200

Cal 4

500
 
q.s. to 10 ml

500


RBC matrix was prepared using left-over blood submitted for immunosuppressant testing. DHAP concentration in this matrix was <10 μM




 


6.

Quality controls (QCs) are made according to Table 2 using standards (see Note 1 ).


Table 2
Preparation of quality controls

































 
2°(μl)
3° (μl)
RBC matrix
Final conc. (μM)

QC 1
  
200
q.s. to 10 ml
 20

QC 2
150
  
q.s. to 10 ml
150

QC 3
400
  
q.s. to 10 ml
400


RBC matrix was prepared using left-over blood submitted for immunosuppressant testing. DHAP concentration in this matrix was <10 μM

 





2.4 Analytical Equipment and Supplies




1.

Triple TOF™5600 (AB Sciex).

 

2.

Acuity UPLC (Waters).

 

3.

Column: Agilent Eclipse XDB, C8, 3.5 μM, 4.6 × 150 mm.

 



3 Methods



3.1 Stepwise Procedure




1.

Pipette 100 μl calibrators, patient sample hemolysate or controls to 1.5 ml microcentrifuge tubes.

 

2.

Add 200 μl protein precipitation reagent and vortex for 1 min.

 

3.

Centrifuge for 10 min at 12,000 × g.

 

4.

Transfer 100 μl supernatants to autosampler vials.

 

5.

Inject into 10 μl into the HPLC /TOF-MS.

 

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Oct 21, 2016 | Posted by in BIOCHEMISTRY | Comments Off on Quantification of Dihydroxyacetone Phosphate (DHAP) in Human Red Blood Cells by HPLC-TripleTOF 5600™ Mass Spectrometer

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