Table 1.

Multi-stepwise gradient elution program and multiple reaction monitoring (MRM) transitions and their associated mass spectrometric parameters for analysis of target analytes.

Gradient elution program				MRM and mass spectrometric parameters
Time, min	(A), %	(B), %	Flow rate, mL/min	Analyte	RT, min	Ion type	Ion transition	CE, eV	Dwell time, ms	Fragmentor, V
0.01	95	5	0.75	3OH-COT	3.1	Q	193.1 → 80.0	28	175	115
3.00	85	15	0.75			C	193.1 → 58.1	36	175	115
5.00	70	30	0.75			L	196.1 → 80.1	28	175	115
6.00	15	85	0.75	COT	4.2	Q	177.1 → 80.1	20	175	115
8.00	15	85	0.75			C	177.1 → 98.0	20	175	115
9.00	0	100	1.00			L	180.1 → 80.1	28	175	120
11.00	0	100	1.00	COOH-THC	8.1	Q	343.4 → 299	17	175	204
12.00	95	5	1.00			C	343.4 → 245	29	175	204
13.00	95	5	1.00			L	352.5 → 308	21	175	204

Note: (A) = 6.5 mM ammonium acetate (pH 5.1), (B) = acetonitrile, RT = retention time, CE = collision energy, 3OH-COT = trans-3′-hydroxycotinine, COT = cotinine, COOH-THC = 11-nor-9-carboxy-Δ9-tetrahydrocannabinol, Q = quantification ion, C = confirmation ion, L = labeled ion. The mass spectrometer divert valve position was switched to waste during the following time segments: 0–2.6 min, 5.0–7.5 min, 8.5–13 min.