Application Note 271 Quantitative Analysis of Nitrogen Mustard Hydrolysis Products as Ethanolamines INTRODUCTION Reported methods for ethanolamines analysis include Ethanolamines have been used as bio- and GC or LC separation with MS detection.8 The GC-MS environmental markers for nitrogen mustards (HN1, HN2, methods involve labor-intensive derivatization which and HN3), which are listed on the Chemical Weapons limits throughput, and reported LC methods usually suffer Convention Schedule of Chemicals1 to monitor potential from poor retention and chromatographic separation with exposures. Direct quantification of exposure to HN1, reversed-phase (RP) columns. A fast LC-MS/MS method HN2, and HN3 is difficult due to their reactivity, extent reported the total separation of MDEA, EDEA, and TEA.6 of metabolism, and short half-life.2 Nitrogen mustards However, the estimated retention factor (k) for the first- readily react with biomolecules and are found in urine eluted TEA was less than one, making the method subject as the hydrolysis products: N-methyldiethanolamine to possible interference from sample matrices, which was (MDEA), N-ethyldiethanolamine (EDEA), and confirmed in the same report. triethanolamine (TEA).3 This study reports a rapid separation liquid Over half a million tons of ethanolamines are chromatography (RSLC) tandem mass spectrometric produced annually and used as emulsifying agents, (MS/MS) method for quantitative analysis of ethanolamines detergents, ingredients in bactericides and cosmetics, and in environmental water samples. An Acclaim® Trinity™ P1 also in the pesticide manufacturing process.4 Inefficient Mixed-Mode column featuring reversed-phase, anion- removal and/or inappropriate disposal of ethanolamimes exchange, and cation-exchange retention mechanisms was may cause adverse effects to the environment. used to provide retention and resolution for all analytes To monitor human and environmental exposure to within 5 min. The MS detector was operated in multiple nitrogen mustard, and also the removal of ethanolamines reaction monitoring (MRM) mode, and an isotope labeled from industrial discharged waste, a quantitative analytical internal standard (IStd) was used to provide selective and method is desired. sensitive detection and to ensure quantification accuracy. EQUIPMENT MASS SPECTROMETRIC CONDITIONS Dionex UltiMate® 3000 RSLC system including: System: Triple quadropole mass DGP-3600RS dual gradient pump spectrometer with ESI WPS-3000TRS autosampler Curtain Gas (CUR): 15 psi TCC-3200RS column oven Collision Gas (CAD): Medium IonSpray Voltage (IS): 4500 V CONDITIONS Temperature (TEM): 700 °C Column: Acclaim Trinity P1 (2.1 × 100 mm, Ion Source Gas 1 (GS1): 50 psi 3 µm, P/N 071389) Ion Source Gas 2 (GS2): 20 psi Mobile Phase: Isocratic, 90% Acetonitrile; 5% DI water; Ihe: On 5% Ammonium formate 100 mM, pH 3.7 Acquisition Mode: Multiple reaction monitoring Flow Rate: 0.6 mL/min (MRM); refer to Table 1 for Temperature: 20 °C details on MRM scan parameters Inj. Volume: 20 µL Software: DCMSLink™,a Chromeleon®- based software module providing the interface for controlling a wide range of Dionex chromatography instruments from different mass spectrometer software platforms. Table 1. MRM Scan Parameters of Studied Analytes Q1 Q3 DP CE CXP tR Peak No. Analyte ID Time (ms) (m/z) (m/z) (V) (V) (V) (min) EDEA-1 134.1 116.0 75 51 21 8 1 N-ethyldiethanolamine 1.8 EDEA-2 134.1 72.0 25 51 25 4 MDEA-1 120.1 102.0 75 46 19 8 2 N-methyldiethanolamine 2.3 MDEA-2 120.1 58.0 25 46 27 4 TEA-1 150.0 132.0 75 61 19 10 3 Triethanolamine 2.7 TEA-2 150.0 88.0 25 61 23 6 DEA-1 106.1 88.0 350 66 19 6 4 Diethanolamine 3.6 DEA-2 106.1 70.0 50 66 21 4 5 Diethanolamine-d8 DEA-IS 114.1 78.0 100 53 24 6 3.6 EA-1 62.0 44.1 350 46 15 6 6 Ethanolamine 4.0 EA-2 62.0 45.0 50 46 19 6 The 1st MRM of each analyte was used for quantiation, and the 2nd MRM was used for confirmation only. 2 Quantitative Analysis of Nitrogen Mustard Hydrolysis Products as Ethanolamines HO OH HO D OH OH D D D N D D H N H N 2 D H D Ethanolamine (EA) Diethanolamine (DEA) Diethanolamine-d8 (DEA-IS) CAS: 141-43-5 CAS: 111-42-2 CAS: 103691-51-6 Chemical Formula: C H NO Chemical Formula: C2H7NO 4 11 2 Chemical Formula: C4H3D8NO2 Exact Mass: 61.05 Exact Mass: 105.08 Exact Mass: 113.13 Molecular Weight: 61.08 Molecular Weight: 105.14 Molecular Weight: 113.18 HO OH HO OH HO OH N N N OH N-Methyldiethanolamine (MDEA) N-Ethyldiethanolamine (EDEA) Triethanolamine (TEA) CAS: 105-59-9 CAS: 139-87-7 CAS: 102-71-6 Chemical Formula: C5H13NO2 Chemical Formula: C6H15NO2 Chemical Formula: C6H15NO3 Exact Mass: 119.09 Exact Mass: 133.11 Exact Mass: 149.11 Molecular Weight: 119.16 Molecular Weight: 133.19 Molecular Weight: 149.19 27914 Figure 1. Chemical structures of studied compounds. PREPARATION OF SOLUTIONS AND REAGENTS All chemicals were dissolved in deionized (DI) Chemical and Reagents water to prepare individual primary stock solutions Standards of studied analytes were purchased from at 1000 µg/mL (ppm). Working stock solutions were Sigma-Aldrich: ethanolamine (EA, CAS: 141-43-5, prepared for each analyte by diluting primary stock Aldrich: 411000), diethanolamine (DEA, CAS: 111-42-2, solutions in DI water to 1 ppm, 100 ppb, 10 ppb, and Fluka: 31589), N-methyldiethanolamine (MDEA, CAS: 1 ppb to prepare calibration standards. A working stock 105-59-9, Aldrich: 471828), N-ethyldiethanolamine solution for the internal standard was prepared at (EDEA, CAS: 139-87-7 Aldrich: 112062), triethanolamine 100 ppb in deionized water for the preparation of (TEA, CAS: 102-71-6, Fluka: 90279). Isotope labeled calibration standards and to spike unknown samples. Calibration standards were prepared in DI water at 8 internal standard (IStd) diethanolamine-d8 (DEA-IS) was purchased from C/D/N Isotopes (CAS: 103691-51-6, levels: 0.05 ppb, 0.1 ppb, 0.5 ppb, 1 ppb, 2 ppb, 5 ppb, D-5308). Figure 1 shows the chemical structures and 10 ppb, and 20 ppb. Each level contains all five target related information. analytes with internal standard spiked at 1 ppb. Ammonium formate was purchased from Aldrich (516961). Acetonitrile was obtained from Burdick & Sample Preparation Jackson (HPLC grade, AH015-4). Deionized water Surface water samples were collected in HDPE (18.2 MΩ-cm resistance) used in this study was obtained plastic bottles and stored under refrigeration at 4 °C from a Millipore water station. until analysis. An aliquot of each water sample was spiked with internal standard at 1 ppb in a 1.5 mL autosampler vial and analyzed directly (filter the surface water samples when necessary, e.g., if suspended particles are observed). Application Note 271 3 RESultS AND DISCUSSION EDEA Chromatography 3.4e4 S/N = 598 Intensity, As shown in Figure 2, all five target analytes were 134 11 6 cps - Noise - separated to baseline within 4.5 min. A retention factor 2.9e4 MDEA S/N = 353 (k') of 3.3 for the first-eluted EDEA indicated sufficient Intensity, cps 120 102 retention for all analytes and thus ensured the separation - Noise - 2.9e4 TEA of targeted analytes from early eluting species. Different Intensity, S/N = 204 cps from general RP columns, the Acclaim Trinity P1 - Noise - 0.48 150 132 Mixed-Mode column features RP and ion exchange 1.5e4 DEA-IS S/N = 491 Intensity, mechanisms, thus providing unique selectivity for cps - Noise - 114 78 ionizable organics. For the mixed-mode column, eluent 5.8e3 DEA S/N = 178 strength is affected by organic modifier composition, Intensity, cps - Noise - 106 88 buffer type, buffer pH, and buffer concentration. Refer 9.7e2 EA S/N = 42 to the column manual for more information on method Intensity, 62 44 development and modification. The conditions described cps - Noise - 4.2e4 EDEA in the experimental section were developed to achieve MDEA TEA Quantitative MRM Intensity, sufficient retention and total resolution for all target DEA-IS cps analytes with consideration of method throughput. DEA EA 0 Although the separation was completed within 4.5 min, 0 1234586 7 the total run time was set at 8 min to elute any Minutes 27915 possible strongly retained species and thus improve method ruggedness. Figure 2. MRM chromatograms of five ethanolamines by RSLC-MS/MS on an Acclaim Trinity P1 column with 0.5 ppb of each analyte. Mass Spectrometry The aim of this study was to develop a selective and sensitive method for the direct analysis of trace The two final MRM transitions were selected that level ethanolamines in environmental water samples, showed specific MS peaks with better intensity. It is worth therefore, MS/MS instrumentation was selected for its noting that interference was observed for both DEA MRM sensitivity and ability to provide trace level detection. channels from TEA and EDEA; and this can be explained In addition, the selectivity of MS/MS instrumentation by the source region fragmentation of TEA and EDEA: + + allows minimal sample preparation and cleanups. The [M-C2H3OH+H] , and [M-C2H4+H] , respectively, which MS/MS instrument was tuned and run in MRM mode. have the identical m/z as the precursor ion of DEA at With continuous infusing of individual standards, each 106 m/z. This observation also indicated that target analyte showed a strong protonated molecular ion chromatographic separation for EDEA, TEA, and DEA [M+H]+ in positive ESI mode, and was used as the Q1MS are crucial for quantification accuracy. The scan time precursor ion for MRM experiments.