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A new retention index system for LC-MS
Quilliam, Michael Arthur; Békri, Khalida; Mcnamara, Caitlin; Giddings, Sabrina; Hui, Joseph
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Measurement Science and Standards
Michael A. Quilliam, Khalida Békri, Caitlin McNamara, Sabrina Giddings, Joseph Hui
Biotoxin Metrology, National Research Council, Halifax Canada
Conclusions
A unique set of homologous compounds, N-alkylpyridine-3-sulfonates, has been prepared for the measurement of retention indices in LC-MS and LC-UV. This RI system should be useful in databases for compound identification. It will also be useful for establishing scheduled SRM windows for LC-MS methods.
References
1. Smith, R.M., Retention index scales used in high-performance liquid chromatography. J. Chromatography Library 57, 93-144 (1995).
2. Quilliam, M.A., Retention index standards for liquid chromatography, Patent WO2013/134862A1.
Figure 7. xxxxx
Applications
Application of the RI system to microcystins (cyclic peptides produced by cyanobacteria) and an assortment of veterinary drugs used in aquaculture are shown in Figures 7 and 8 and in Tables 1 and 2, respectively. Good repeatability within day and between days was observed.
One interesting application of this data is the prediction of retention time windows for scheduled SRM on different LC-MS systems, where RTs can vary significantly.
Table 1: Microcystin RI data from runs with 2 different gradients showing reproducibility. A 2-point correction was applied to the second.
Introduction
Chromatographic retention times (RTs) are invaluable for the identification of analytes in complex samples. Unfortunately, absolute RTs in liquid chromatography (LC) can be highly variable between different laboratories and instruments. Analysis of chemical reference standards is required with each batch of samples to allow a good match of RTs for conclusive identification. Development of modern LC-MS methods based on techniques like scheduled selected reaction monitoring (SRM) also requires the availability of standards to set retention windows and it is tedious and time consuming to establish a method.
A better way to report and catalog retention data is to use a retention index (RI) system. RI values are determined by measuring analytes’ RTs relative to those of a series of homologous reference compounds. Ideally sample and RI standards should be co-injected. Several RI systems (Fig. 1) have been reported for use in LC-UV [1]. However, none of these are well suited to electrospray ionization (ESI) LC-MS as they exhibit low sensitivity. In addition, some of them are not suitable for the low RT range where polar compounds elute.
Project Objectives
The following characteristics were selected for the new standards: • High sensitivity in both positive and negative ion ESI-MS.
• Common fragment ion to facilitate MS detection. • A strong UV chromophore for use in LC-UVD.
• Overall neutral charge state independent of mobile phase pH. • A homologous series spanning the widest RPLC gradient run.
The N-alkylpyrinium-3-sulfonate (NAPS) standards (Fig. 1) [2] have been designed with two permanently ionized functions (quaternary amine and sulfonate), which enhance detectability in mass spectrometry in both the positive and negative ion modes. They have an overall neutral charge state that will not vary with changes in the mobile phase pH, thus making their RTs less sensitive to pH changes in the mobile phase. The compounds also have a good UV chromophore with an absorbance maximum at 265 nm.
Experimental
• Synthesis: reaction of alkyl halides with pyridinium-3-sulfonate.
• Preparation of reference material (RM-RILC): 20 standards at 100 µM in
CH3OH; packaged into flame-sealed glass ampoules.
• Use of RM: RM-RILC can be mixed or co-injected with a sample at 1 to 10% of
total volume injected.
• LC-UV: Agilent 1290 system with DAD at 265 nm.
• LC-MS: Sciex QTRAP 4000 with ESI and Thermo Exactive Orbitrap with ESI or
APCI sources. SRM used for Sciex; alternating full and HCD scans used for Orbitrap.
• Columns: 2 mm id x 5 or 15 cm packed with (a) 2.7 µm Poroshell SB-C18; (b)
1.8 µm Zorbax SB-C18; (c) 3 µm Hypersil BDS-C8; (d) 2.5 µm Luna C18(2)-HST; (e) 2.5 µm Synergi Hydro RP.
• Mobile phases: 0.25 mL/min CH3CN/H2O + 50 mM HCOOH/2 mM HCOONH4
(pH 3). Different pHs and CH3OH also tested.
• Curve fitting: SRS1 SoftwareLLC, http://www.srs1software.com (Excel macro)
Figure 1. Retention index standards reported previously for HPLC.
(CH2)nCH3 O O HO Parabens (n = 0 to 10) H3C (CH2)nCH3 O 2-Ketoalkanes (n = 0-21) (CH2)nCH3 O Alkylphenones (n = 0-10) 1-Nitroalkanes (n = 0-9) O HO HO (CH2)nCH3 O 1-[p-(2,3-dihydroxypropoxy)phenyl]-1-alkanones (n = 0 to 5) CH3-(CH2)n-NO2 N (CH2)9CH3 N+ (CH2)9CH3 N+ (CH2)9CH3 H H N H H3C(H2C)8 H SO3 -SO3H SO3 SO3 -N H3C(H2C)8HC SO3 -H or N+ (CH2)9CH3 SO3 - HCOO-C15H25NO3S m/z 299.1555 C15H24NO3S -m/z 298.1482 C15H26NO3S -m/z 300.1638 C16H26NO5S -m/z344.1537 C15H26NO3S+ m/z 300.1627 -HCOOH -CO2 +H +HCOO -N H+ SO3H m/z 160.0063 N H+ OH C5H6NO + m/z 96.0444 N H+ C5H5N•+ m/z 79.0417 SO3 -. m/z 79.9574 N O -C5H4NO -m/z 94.0298 N SO3 -C5H4NO3S -m/z 157.9917 N SO3 -H H C7H8NO3S -m/z 186.0230
Figure 4. Ionization and fragmentation characteristics of the C10-NAPS.
Figure 3. Positive (a,b) and negative (c,d) spectra of the C10-NAPS acquired
on an Exactive Orbitrap MS with full (a,c) or HCD (b,d) scans.
8.0e+6 m/z 50 100 150 200 250 300 350 7.0e+5 1.6e+6 1.6e+7 [M+H]+ 300.1626 [M+Na]+ 322.1450 160.0063 79.0421 96.0447 [M+H]+ 300.1626 [M+HCOO]-344.1535 [M+HCOO-CO2] -300.1640 [M-H]-298.1483 SO3 -79.9564 94.0290 157.9910 a b c d
A New Retention Index System for LC-MS
Mass Spectral Characteristics
• Abundant signals in both positive and negative ion ESI. In
positive, the molecule is protonated to form an [M+H]+ ion. In
negative, a formate attachment ion, [M+HCOO]-, is formed.
• Collision-induced dissociation gives common ions at m/z 160 in positive mode and m/z 80 in negative mode. These can be
created by using a high orifice potential or a collision cell.
LC-MS and LC-UV Characteristics
• Excellent behavior in gradient elution reversed phase LC.
• Easily detected by MS or UV absorbance (265 nm). An injection of 10 to 100 pmol is sufficient (0.1 to 1 µL of the RM).
• Triple quadrupole analysis can be achieved with SRM using individual transitions or with a high orifice voltage plus a 160 > 160 transition at low collision energy.
• Orbitrap LC-MS analysis best achieved with alternating full scans and HCD scans, the latter allowing extraction of an m/z 160 mass chromatogram.
• A plot of RT vs. RI value (100 x carbon length) and a cubic spline fit results in a calibration curve such as that shown in Fig. 5c.
(a) Short gradient (25-75%B, 30 min) (b) Long gradient (25-75%B, 60 min) 2-point correction
Microcystin Precursor Average 1 day SD 4 day SD Average 1 day SD 4 day SD of (b) to match (a) Code Ion > m/z 135 RT (min) RI(a) (n=5) (n=5,d=4) RT (min) RI(b) (n=5) (n=5,d=4) RI(c) % Diff. to (a)
3dm7dmRR 505.8 9.17 832.6 0.4 0.5 11.33 842.3 0.7 1.2 831.6 -0.11 RR 519.8 9.64 847.2 0.3 0.8 12.18 858.4 0.5 1.5 847.2 0.00 7dmRR 512.8 9.72 850.4 0.4 0.7 12.32 862.0 0.3 1.2 850.8 0.04 Nod-R 825.5 10.91 888.7 0.5 0.7 14.29 901.3 0.2 1.1 888.9 0.03 YR 1045.6 12.12 929.3 0.4 0.6 16.75 947.0 0.4 0.8 933.3 0.42 7dmYR 1031.6 12.30 934.1 0.4 0.7 17.01 952.1 0.5 0.8 938.3 0.44 LR 995.6 12.69 946.7 0.1 1.3 17.75 964.9 0.2 1.6 950.7 0.41 7dmLR 981.6 12.87 953.3 0.3 1.3 18.19 972.9 0.2 1.9 958.4 0.53 3dm7dmLR 967.6 13.24 965.6 0.4 1.2 18.85 984.7 0.5 1.3 969.9 0.43 (O-Me)LR 1009.9 13.61 977.8 0.5 1.3 19.59 997.6 0.4 1.5 982.4 0.46 7dmHilR 995.6 13.82 985.2 0.8 0.9 19.94 1004.5 0.5 1.4 989.0 0.38 RA 953.8 14.16 995.3 0.6 2.1 20.45 1012.8 0.2 2.3 997.1 0.18 LA 910.6 16.93 1092.5 0.2 2.4 25.63 1110.0 0.4 2.8 1091.0 -0.13 isoLA 910.6 17.35 1106.8 0.3 2.9 26.39 1124.4 0.2 3.0 1104.8 -0.18 LY 1002.9 17.91 1128.3 0.4 1.8 27.68 1150.4 0.2 1.9 1129.9 0.14 LW 1025.9 20.77 1232.9 0.2 1.8 33.20 1259.1 0.5 1.9 1234.5 0.12 LF 986.8 21.38 1255.1 0.3 2.4 34.25 1280.6 0.4 2.5 1255.1 0.00
Drug Class Name of Compound Abbrev. SRM transition RI
SD (n = 5)
Sulfonamides Sulfanilamide SNA 173.1 > 92.1 379.7 1.3
Sulfacetamide SAA 215.1 > 156.1 471.9 0.6 Sulfadiazine SDZ 251.1 > 156.1 500.1 0.4 Sulfathiazole STZ 256.1 > 156.1 530.4 0.4 Sulfapyridine SPR 250.1 > 156.1 531.9 0.2 Sulfamerazine SMR 265.1 > 92.1 546.8 0.4 Sulfamethazzine SMZ 279.1 > 186.1 584.0 0.3 Sulfamethizole SML 271.1 > 156.1 600.3 0.2 Sulfamethoxypyridazine SMP 281.1 > 126.1 602.9 0.2 Sulfachloropyridazine SCP 285.1 > 156.1 653.9 0.3 Sulfadoxine SDO 311.1 > 156.1 676.9 0.3 Sulfamethoxazole SMO 254.1 > 156.1 685.1 0.2 Sulfaquinoxaline SQO 301.1 > 156.1 780.4 0.2
Nitroimidazoles 1-(2-Hydroxyethyl)-2-hydroxymethyl-5-nitroimidazole MNZ-OH 188.1 > 123.1 419.5 0.5
2-Hydroxymethyl-1-methyl-5-nitrimidazole HMMNI 158.1 > 140.1 453.1 0.2 Metronidazole MNZ 172.1 > 128.1 457.3 0.5 Dimetridazole DMZ 142.1 > 96.1 482.6 0.3 Ronidazole RNZ 201.1 > 55.1 493.3 0.4 1-Methyl-2-(2'-hydroxyisopropyl)-5-nitroimidazole IPZ-OH 186.1 > 168.1 590.6 0.2 Ipronidazole IPZ 170.1 > 109.1 676.9 0.4
Fluoroquinolones Ciprofloxacin CIPRO 332.2 > 245.1 601.0 0.2
Danofloxacin DANO 358.2 > 96.1 614.1 0.3
Enrofloxacin ENRO 360.2 > 316.2 625.2 0.4
Sarafloxacin SARA 386.2 > 342.2 666.2 0.3
Quinolones Oxolinic acid OXA 262.1 > 216.1 744.6 0.6
Macrolides Erythromycin ERY 734.5 > 158.1 833.3 0.6
Dyes Leucocrystal Violet LCV 374.4 > 358.1 660.3 0.6
Leucomalachite green LMG 331.1 > 316.1 983.2 0.9
Malachite Green MG 329.2 > 313.1 1180.8 1.2
Crystal Violet CV 372.4 > 356.1 1406.5 1.1
Brilliant Green BG 385.3 > 341.1 1572.6 1.0
Figure 8. LC-MS measurement of RI values for a mixture of veterinary drugs.
Time (min) 5 10 15 20 25 30 DMZ HMMNI IPZ MNZ SNA IPZOH MNZOH RNZ SAA SPR SDZ SMO STZ OXA SMR SML SMZ SMP SCP SQO SDO MG LMG CIPRO DANO ENRO CV LCV SARA ERY BG 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800
Figure 6. Effect of column length and gradient on the RI calibration curves.
Retention Index 400 600 800 1000 1200 1400 1600 1800 T ime ( mi n ) 0 5 10 15 20 25 30 Retention Index 400 600 800 1000 1200 1400 1600 1800 0 10 20 30 40 50 60 150 x 2 mm Poroshell 25-75% in 60 min 150 x 2 mm Poroshell, 25-75% in 30 min 150 x 2 mm Poroshell, 5-100% in 30 min 50 x 2 mm Poroshell, 5-100% in 30 min
Effect of LC Parameters
Parameters tested: (a) column length; (b) LC dead volume; (c) flow rate; (d) gradient start/stop % and rate; (e) stationary phase; and (f) mobile phase solvents, buffers and pH. The effects of column length and gradient on the RI calibration curves are shown in Fig. 6. RI values for analytes were very repeatable under each condition. Some variation did occur with a change of conditions, especially with different mobile and stationary phases as expected. A two-point correction using internal standards belonging to a class of analyte can help correct for these shifts (see Table 1).
Time (min) 8 10 12 14 16 18 20 22 505>135 519>135 512>135 825>135 1045>135 1031>135 995>135 981>135 967>135 1009>135 953>135 910>135 1002>135 1025>135 986>135 800 900 1000 1100 1200 1300
Figure 7. LC-MS measurement of RI values for a mixture of microcystins.
Table 2: RI data for veterinary drugs used in aquaculture.
Figure 5. Analysis of RM-RILC by
Orbitrap LC-MS (a) and LC-UV (b) using a 150 x 2 mm, 2.7µm Poroshell SB-C18, 5-100%
CH3CN in 30 min, 0.25 mL/min
50 mM HCOOH + 2 mM
HCOONH4 (* = gradient ghost
peaks). A plot of RT vs RI value is shown in (c). A cubic spline fit results in a calibration curve
Retention Index 200 400 600 800 1000 1200 1400 1600 1800 2000 T im e ( m in) 0 5 10 15 20 25 30 0 5 10 15 20 25 30 T ot al I on C ur rent 2e+8 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 100 200 * a b c
cubic spline fit
Figure 2.
The new RI standards.
Time (min) 0 5 10 15 20 25 30 A bs or banc e at 265 nm ( m A U ) 0 20 40 60 80 100 120 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 100,200 N+ SO3 (CH2)nCH3 n = 0-19 N-Alkylpyridinium-3-sulfonates (NAPS)
