Table 8.

Troubleshooting table

Problem	Possible reason(s)	Solution(s)
Low DNA yields	→ Incomplete lysis of tissue or cells	→ Optimize cell or tissue disruption conditions (e.g., increase time or intensity of mechanical homogenization)
	→ DNA yield is sometimes tissue dependent, some tissues yield higher DNA amount than others	→ Increase sample size
RNA contamination	→ Inadequate RNA degradation during DNA isolation	→ Increase RNase during extraction and prolong the incubation time
Inconsistent retention times during HPLC pre-purification or LC/MS-MS	→ Column not equilibrated prior to purification	→ Increase column equilibration time
	→ Inconsistent column pressures due to inadequate degassing of mobile phases	→ Use a degassing apparatus on the HPLC or Sonicate buffers for 20 min
	→ Problems with the stationary phase in the column (e.g., use of high aqueous isocratic conditions during LC/MS/MS analysis leads to column collapse)	→ Regenerate column by running 100% acetonitrile overnight at 0.100 ml/min; replace column, if necessary
dI levels are higher than expected	→ Deaminase contamination	→ Include or increase coformycin during all stages of DNA isolation and processing
	→ Contamination of dI with dA during HPLC pre-purification or LC/MS-MS; m/z values differ by 1, so dA isotopomer signals (M+1) contribute to dI signal	→ Decrease the % organic solvent to resolve the dA and dI peaks
dX and dO levels are higher than expected	→ Contamination of dX or dO with dG during HPLC pre-purification or LC/MS-MS; m/z values differ by 1, so dG isotopomer signals (M+1) contribute to dX/dO signal	→ Decrease the % organic solvent to resolve the dG and dX or dO peaks
8-Oxo-dG levels are higher than expected	→ Artifactual formation during sample work-up or in vacuo drying	→ Include desferrioxamine and butylated hydroxytoluene during cell processing, DNA extraction and hydrolysis steps; process samples on ice; minimize sample evaporation time
	→ As 8-oxo-dG can be formed from 2′-deoxyguanosine (dG) in the ion source of the mass spectrometer it is important that dG contamination separates well from 8-oxo-dG prior to entering the MS	→ Decrease the % organic solvent to resolve dG from 8-oxo-dG
εdC levels are higher than expected	→ Contamination of εdC with dA due to similar HPLC retention times and identical m/z values	→ Optimize the separation of εdC and dA in the pre-purification step. Also, optimize the HPLC step of the LC/MS-MS analysis to such that εdC elutes 2-3 min before dA.