TABLE 2

Overview of structural information obtained by LC-MSⁿ for bendamustine metabolites detected in human urine

Peak Identification	T_R^{^a}	T_R^{^b}	Proposed Elemental Composition	[M + H]⁺		Δppm	Major or Specific Product Ions [M + H]^{+ ^c}	Proposed Metabolic Conversions at:
Peak Identification	T_R^{^a}	T_R^{^b}	Proposed Elemental Composition	Theoretical	Observed	Δppm	Major or Specific Product Ions [M + H]^{+ ^c}	Chloroethyl Moiety 1	Chloroethyl Moiety 2	Benzimidazole/Butyric Acid Moiety
	min
1		3–5^{^d}	Unknown					Unknown	Unknown	Unknown
2	14.7	7.9	C₁₆H₂₄N₃O₈P	418.1374	418.1363	−2.63	338.1705 (g), 320.1600 (g + f), 302.1495 (g + 2f)	Phosphate conjugation	Hydrolysis	γ-Hydroxylation
3	18.7	10.9	C₁₅H₂₁N₃O₄	308.1605	308.16	−1.62	263.1260 (c1), 214.0971 (c1 + b2 + f)	Hydrolysis	Hydrolysis	N-Demethylation
4	19.7	11.0	C₁₉H₂₈N₄O₆S	441.1802	441.179	−2.72	352.1320 (h), 294.1444 (c1), 276.1338 (c1 + f)	Cysteine conjugation	Hydrolysis	γ-Hydroxylation
5	22.0	12.1	C₁₆H₂₄N₃O₇P	402.1425	402.1415	−2.49	322.1756 (g), 304.1650 (g + f)	Phosphate conjugation	Hydrolysis
6	23.8	13.3	C₁₆H₂₃N₃O₅	338.1710	338.1704	−1.77	302.1496 (2f), 276.1703 (e + f)	Hydrolysis	Hydrolysis	γ-Hydroxylation
7	24.2	13.9	C₂₀H₂₈N₆O₄	417.2245	417.2234	−2.64	304.1651 (a1), 278.1495 (c1)	Creatinine conjugation	Hydrolysis
8	29.0	15.8	C₁₉H₂₈N₄O₅S	425.1853	425.1841	−2.82	336.1371 (h), 278.1494 (c1), 260.1389 (c1 + f)	Cysteine conjugation	Hydrolysis
9	29.4	16.9	C₁₄H₁₇N₃O₃	276.1343	276.1336	−2.53	258.1232 (f), 230.1283 (e), 217.0967 (d)	N-Dealkylation	Oxidation
10 (HP2)	33.7	17.6	C₁₆H₂₃N₃O₄	322.1761	322.1754	−2.17	304.1653 (f), 277.1418 (c1), 246.1234 (c1 + b2)	Hydrolysis	Hydrolysis
11	40.7	18.9	C₂₁H₂₅N₇O₆	472.1939	472.1923	−3.39	429.1871 (i), 411.1766 (i + f), 385.1975 (i + j), 304.1651 (a1), 278.1494 (c1), 260.1389 (c1 + f)	Uric acid conjugation	Hydrolysis
12	50.2	19.9	C₂₀H₂₇N₃O₉	454.1820	454.1805	−3.30	408.1757 (k), 304.1651 (a1), 286.1545 (a1 + f)	Conjugation with 150.0154 Da	Hydrolysis
13	54.6	23.1	C₂₀H₂₇N₆O₃Cl	435.1906	435.1894	−2.76	322.1311 (a1), 296.1155 (c1)	Creatinine conjugation
14	57.3	25.4	C₁₄H₁₈N₃O₃Cl	312.1109	312.1103	−1.92	276.0894 (2f), 248.0945 (e + f)	N-Dealkylation		γ-Hydroxylation
15	64.4	24.2	C₁₆H₂₃N₃O₃S	338.1533	338.1525	−2.37	278.1494 (c1), 260.1388 (c1 + f)	Thiol conjugation	Hydrolysis
16	70.7	26.1	C₁₆H₂₃N₃O₆PCl	420.1086	420.1076	−2.38	340.1418 (g), 322.1313 (g + f), 304.1207 (g + 2f)	Phosphate conjugation
17	71.1	25.9	C₂₃H₃₆N₄O₅Cl	483.2363^{^e}	483.2353^{^e}	−2.07	424.1624 (l), 354.1572 (m), 336.1468 (m + f), 322.1311 (a1), 304.1206 (a1 + f)	Hydrolysis		Conjugation with 161.1042 Da
18	71.3	27.1	C₁₆H₂₂N₃O₄Cl	356.1372	356.1362	−2.81	320.1155 (2f), 294.1363 (e)	Hydrolysis		γ-Hydroxylation
19	72.5	28.6	C₁₄H₁₈N₃O₂Cl	296.1160	296.1153	−2.36	278.1049 (f), 250.1100 (e), 242.1283 (a2 + f)	N-Dealkylation
20	73.7	26.5	C₁₉H₂₇N₄O₄SCl	443.1514	443.1503	−2.48	354.1034 (h), 296.1157 (c1), 278.1052 (c1 + f)	Cysteine conjugation
21 (HP1)	82.7	29.2	C₁₆H₂₂N₃O₃Cl	340.1422	340.1416	−1.76	322.1314 (f), 286.1547 (a2 + f), 246.1234 (c1 + b2)	Hydrolysis
22	97.6	32.2	C₂₁H₂₉N₄O₅SCl	485.1620	485.1605	−3.09	356.1187 (n), 296.1155 (c1), 278.1049 (c1 + f)	N-Acetylcysteine conjugation
23	99.4	35.1	C₁₆H₂₂N₃O₂SCl	356.1194	356.119	−1.12	302.1320 (a2 + f), 296.1160 (c1)	Thiol conjugation
24 (M3)	104.5	35.1	C₁₆H₂₁N₃O₃Cl₂	374.1033	374.1023	−2.67	338.0816 (2f), 310.0868 (e + f)			γ-Hydroxylation
25 (M4)	105.1	35.3	C₁₅H₁₉N₃O₂Cl₂	344.0927	344.0919	−2.32	326.0815 (f), 298.0867 (e), 290.1049 (a1 + f)			N-Demethylation
26 (BM)	109.2	36.3	C₁₆H₂₁N₃O₂Cl₂	358.1084	358.1076	−2.23	304.1206 (a1 + f), 228.1127 (c1 + b2 + f)

BM, bendamustine.
↵a Retention time with the 180-min HPLC method.
↵b Retention time with the 60-min HPLC method.
↵c The most abundant product ions are displayed in bold. The letters in parentheses indicate the proposed fragmentation. The letters a1, a2, b1, b2, c1, c2, d, and e correspond to the fragmentations indicated with these letters in Table 1. The other letters have the following proposed fragmentations: f, loss of H₂O; g, loss of HPO₃; h, loss of alanine; i, loss of OCNH; j, loss of CO; k, loss of HCOOH; l, loss of C₃H₉N; m, loss of C₆H₁₁NO₂; n, loss of N-acetyl-2-iminopropionic acid.
↵d Retention time in radiochromatogram.
↵e [M]⁺ instead of [M + H]⁺ value if conjugate is carnitine as hypothesized under Discussion.