Almost all the classes have RSD (%) below 20% except for C15:0 CoA with slightly higher value of 22.9% at low level. The recovery was in the range of (53-123) % for all standards. It was observed that recovery of long-chain acyl-CoA species is slightly lower. The reason for this may be that long-chain acyl-CoA species have lower polarity compared to short-and medium-chain species, which could result in their migration to the non-polar lower layer. The matrix effect was in the range of (85-133)%. The carryover was analyzed in the blank samples placed right after the highest calibration point in HepG2 cells before extraction and was below 1% for all standards. We are using non-endogenous compounds as internal standards based on the chain length of endogenous targets. These standards elute in close proximity to the endogenous compounds present in the sample. Hence, the issues related to poor recovery, ion suppression and matrix effects can be compensated as internal standards and endogenous compounds will parallelly go through the same processing.