Table 2.

Method validation data using different internal standard options. Accuracy is expressed as the percent measurement in comparison to true concentration. Precision is shown as the percent CV for replicate analyses over three days of measurement. The SIL-VVV extended refers to the extended peptide, digest refers to adding the internal standard prior to digestion and quench to adding the internal standard after trypsin digestion. For RDH11, the mid-QC was prepared as a pooled sample of human liver microsomes from four donors.

Internal Standard	QC (low)		QC (mid)		QC (high)		r2 for calibra- tion curve
	Accuracy (%)	CV%	Accuracy (%)	CV%	Accuracy (%)	CV%
RDH11	22.5 nM		2 mg/mL pooled HLM (n=4)		55 nM
SIL-RDH	89	17		16	81	15	0.97± 0.01
SIL-VVV extended	153	16		12	122	11	0.91± 0.10
SIL-VVV in digestion	120	10		13	113	15	0.96± 0.01
SIL-VVV quench	98	8		8	94	8	0.98± 0.01
ALDH1A1	150 nM		325 nM		600 nM
SIL-ANN-1A1 in digestion	74	33	93	23	127	20	0.96± 0.02
SIL-ANN-1A1 quench	110	10	111	14	111	11	0.99± 0.01
ALDH1A2	10 nM		32.5 nM		80 nM
ILE in digestion	109	26	120	4	116	16	0.96± 0.02
ILE quench	105	14	92	11	89	15	0.98± 0.01