Retention index standards for liquid chromatography

Abstract

A liquid chromatography method for identifying an analyte of interest utilizing as retention index standards a homologous series of neutrally charged compounds having at least one functional group bearing a positive charge and at least one functional group bearing a negative charge. The method is especially useful for liquid chromatography-mass spectrometry (LC-MS) methods, more especially for LC-MS methods employing electrospray (ESI) or atmospheric pressure chemical ionization (APCI) ionization systems.

Claims

1. A method of identifying an analyte of interest comprising: introducing the analyte of interest together with a series of homologous retention index standards into a liquid chromatography system, the retention index standards comprising neutrally charged compounds, each of the neutrally charged compounds comprising at least one functional group bearing a permanent positive charge and at least one functional group bearing a permanent negative charge; assigning a retention index value to the analyte of interest based on retention times and retention index values of the retention index standards; and, comparing spectral data and the retention index value of the analyte of interest to a library of spectral data and the retention index values for known compounds to identify the analyte of interest.

2. The method according to claim 1, wherein the at least one functional group bearing a positive charge comprises a quaternary amine group.

3. The method according to claim 1, wherein the at least one functional group bearing a negative charge comprises a sulfonate group.

4. The method according to claim 1, wherein the series of homologous retention index standards comprises from 4 to 16 homologues.

5. The method according to claim 1, wherein the series of homologous compounds is co-injected with the analyte of interest into the liquid chromatography system.

6. The method according to claim 1, wherein assigning a retention index value to the analyte of interest comprises interpolating analyte retention times into a fitted curve of a plot of retention time vs. retention index value for the series of homologous retention index standards.

7. The method according to claim 1, wherein the analyte of interest is a toxin, pharmaceutical, drug of abuse, peptide, persistent environmental pollutant, or food contaminant.

8. The method according to claim 1, wherein the liquid chromatography system comprises a liquid chromatography-mass spectrometry system and the spectral data comprises mass spectrometry data.

9. The method according to claim 1, wherein the liquid chromatography system comprises a liquid chromatography system with an ultraviolet absorbance detector and the spectral data comprises ultraviolet absorbance data.

10. The method according to claim 8, wherein assigning a retention index value to the analyte of interest comprises interpolating analyte retention times into a fitted curve of a plot of retention time vs. retention index value for the series of homologous retention index standards.

11. The method according to claim 8, wherein the analyte of interest is a toxin, pharmaceutical, drug of abuse, peptide, persistent environmental pollutant, or food contaminant.

12. The method according to claim 8, wherein the at least one functional group bearing a positive charge comprises a quaternary amine group.

13. The method according to claim 8, wherein the at least one functional group bearing a negative charge comprises a sulfonate group.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In order that the invention may be more clearly understood, embodiments thereof will now be described in detail by way of example, with reference to the accompanying drawings, in which:

(2) FIG. 1 depicts an LC-MS analysis of a 1-alkyl-3-pyridinesulfonic acid (APSA) mixture containing C4 to C18 homologues (compounds of Formula (I) where n=317);

(3) FIG. 2 depicts a spline fit graph for APSA standards with retention times of APSAs plotted against retention index value and fitted with a cubic spline curve;

(4) FIG. 3 depicts ionization and fragmentation characteristics of APSAs;

(5) FIG. 4 depicts LC-MS analysis of a cyanobacterial extract containing microcystins co-injected with a mixture of APSA standards, in which: upper plots show signals for targeted microcystins (not all the analyte peaks labeled) and bottom plot shows signals for the APSA standards (the APSA peaks labeled with their retention index numbers, 400 to 1800);

(6) FIG. 5 depicts a plot of retention time vs. retention index value for APSA standards observed in the LC-MS analysis shown in FIG. 4 (circles and solid trace) and for a separate analysis (triangles and dashed trace) on the same LC column but with a 60 min gradient rather than the 30 min used in FIG. 4;

(7) FIG. 6 depicts LC-MS analysis of a mixture of reference standards of various drugs that are routinely monitored by food inspection agencies because of their use in agriculture and aquaculture;

(8) FIG. 7 depicts a plot of the retention times vs. retention index values for APSA standards observed in the LC-MS analysis shown in FIG. 6, with a cubic spline fit generated to fit the data; and,

(9) FIG. 8 depicts a plot of the retention times vs. retention index values for APSA standards measured in three different LC-MS analyses using two different columns (150 vs. 50 mm length) and two different gradients, with cubic spline fits generated to fit the data.

DESCRIPTION OF PREFERRED EMBODIMENTS

(10) Referring to FIG. 1, an LC-MS analysis of a 1-alkyl-3-pyridinesulfonic acid (APSA) mixture containing C4 to C18 homologues demonstrates that APSAs give good chromatographic performance. With reference to FIG. 2, the plot of retention time vs. retention index for the APSA homologues gives a curve that is well-fitted by a cubic spline. Retention index is defined as the number of carbons in the side chain of the APSA multiplied by 100. The APSA compounds give excellent response in mass spectrometric detection using either positive or negative ion modes. Selected reaction monitoring (SRM) can be used to monitor the compounds as they all fragment to the same product ion (m/z 160 in positive mode and m/z 158 in negative mode) (see FIG. 3). The compounds also have an excellent UV chromophore with an absorbance maximum at 266 nm, so they can be used as retention index markers in LC-UV analyses as well.

Example 1: Use of APSA Retention Index Standards to Identify Microcystins by LC-MS

(11) A sample of cyanobacterial extract containing analytes of interest (i.e. microcystins) was co-injected with a mixture of the C4-C18 APSA standards into a reversed phase liquid chromatography-mass spectrometry system. The system comprises an LC-ESI-QqQ (QTRAP 4000) instrument with an Agilent 2.7 m-Poroshell 120 SB-C18 column (2.1150 mm). Elution conditions comprise mobile phase: A=water, B=MeCN/water (95:5), both with 50 mM formic acid+2 mM ammonium hydroxide at pH 2.2; with a gradient of 25-75% B over 30 min at a hold time of 5 min, flow rate of 0.25 ml/min, and column temperature of 40 C. The liquid chromatography (LC) column provides separation of the complex mixture while the mass spectrometer provides detection of both the microcystins within the sample and the co-injected APSA standards (FIG. 4). The characteristic mass spectral signals and the retention times of the microcystins and APSAs in the sample are measured. In FIG. 4, the top box shows the signals for targeted microcystins, while the bottom box shows signals for the APSA standards. The APSA peaks are labeled with their retention index numbers, 400 to 1800.

(12) An interpolation of microcystin retention time into a fitted curve of the plot of retention time versus retention index value for the APSAs (FIG. 5) results in a measured retention index for the microcystins. In FIG. 5, the plot with circles and solid trace is for the LC-MS analysis shown in FIG. 4 (30 min gradient), while the plot with triangles and dashed trace is a separate analysis on the same LC column under the same conditions but with a 60 min gradient rather than a 30 min gradient. The curve fitting is best done using the method of cubic splines, and FIG. 5 shows the cubic spline fits generated to fit both sets of data.

(13) The resulting retention index of the analyte of interest, as well as its mass spectral data, can then be compared to a database previously established for a wide range of analytes in order to identify the analyte of interest. In the present example, the sample contains more than one analyte of interest (i.e., multiple microcystins) and the method generates a list of analytes identified in the sample (Tables 1a-1c).

(14) TABLE-US-00001 TABLE 1a Short gradient (25-75% B, 30 min) Microcystin Precursor RT Average 1 day SD 4 day SD Code Ion > 135 (min) Rla (n = 5) (n = 5, d = 4) 3dm7dmRR 505.8 9.17 832.6 0.4 0.5 RR 519.8 9.64 847.2 0.3 0.8 7dmRR 512.8 9.72 850.4 0.4 0.7 Nod-R 825.5 10.91 888.7 0.5 0.7 YR 1045.6 12.12 929.3 0.4 0.6 7dmYR 1031.6 12.30 934.1 0.4 0.7 LR 995.6 12.69 946.7 0.1 1.3 7dmLR 981.6 12.87 953.3 0.3 1.3 3dm7dmLR 967.6 13.24 965.6 0.4 1.2 (OMe)LR 1009.9 13.61 977.8 0.5 1.3 7dmHiIR 995.6 13.82 985.2 0.8 0.9 RA 953.8 14.16 995.3 0.6 2.1 LA 910.6 16.93 1092.5 0.2 2.4 isoLA 910.6 17.35 1106.8 0.3 2.9 LY 1002.9 17.91 1128.3 0.4 1.8 LW 1025.9 20.77 1232.9 0.2 1.8 LF 986.8 21.38 1255.1 0.3 2.4

(15) TABLE-US-00002 TABLE 1b Long gradient (25-75% B, 60 min) Microcystin Precursor RT Average 1 day SD 4 day SD Code Ion > 135 (min) Rlb (n = 5) (n = 5, d = 4) 3dm7dmRR 505.8 11.33 842.3 0.7 1.2 RR 519.8 12.18 858.4 0.5 1.5 7dmRR 512.8 12.32 862.0 0.3 1.2 Nod-R 825.5 14.29 901.3 0.2 1.1 YR 1045.6 16.75 947.0 0.4 0.8 7dmYR 1031.6 17.01 952.1 0.5 0.8 LR 995.6 17.75 964.9 0.2 1.6 7dmLR 981.6 18.19 972.9 0.2 1.9 3dm7dmLR 967.6 18.85 984.7 0.5 1.3 (OMe)LR 1009.9 19.59 997.6 0.4 1.5 7dmHiIR 995.6 19.94 1004.5 0.5 1.4 RA 953.8 20.45 1012.8 0.2 2.3 LA 910.6 25.63 1110.0 0.4 2.8 isoLA 910.6 26.39 1124.4 0.2 3.0 LY 1002.9 27.68 1150.4 0.2 1.9 LW 1025.9 33.20 1259.1 0.5 1.9 LF 986.8 34.25 1280.6 0.4 2.5

(16) TABLE-US-00003 TABLE 1c 2-point correction of (b) to match (a) Microcystin Code Precursor Ion > 135 Rlc % Diff. to (a) 3dm7dmRR 505.8 831.6 0.11 RR 519.8 847.2 0.00 7dmRR 512.8 850.8 0.04 Nod-R 825.5 888.9 0.03 YR 1045.6 933.3 0.42 7dmYR 1031.6 938.3 0.44 LR 995.6 950.7 0.41 7dmLR 981.6 958.4 0.53 3dm7dmLR 967.6 969.9 0.43 (OMe)LR 1009.9 982.4 0.46 7dmHiIR 995.6 989.0 0.38 RA 953.8 997.1 0.18 LA 910.6 1091.0 0.13 isoLA 910.6 1104.8 0.18 LY 1002.9 1129.9 0.14 LW 1025.9 1234.5 0.12 LF 986.8 1255.1 0.00

(17) Table 1a provides retention times (RT) and retention indices (RI) measured for the microcystins detected in the LC-MS analysis shown in FIG. 4 (30 min gradient). Excellent reproducibility data for analyses is shown for within-day (number of runs, n=5) and between-day (number of days, d=4) runs. Also shown is data using the same column but with a 60 min gradient (Table 1b). Finally, in Table 1c, the data from the 60 min gradient (Table 1b) has been adjusted to match the 30 min gradient data (Table 1a) using a 2-point correction based upon the retention data for two microcystins, RR and LF. This process allows fine tuning of the matching of data and results in less than 1% difference is observed for the corrected 60 min RI data vs. the 30 min RI data (Table 1c).

Example 2: Use of APSA Retention Index Standards to Establish a Database of Retention Indices of Analytes for LC-MS

(18) A solution containing various reference compounds (i.e. drugs that have been used in agriculture and aquaculture and that are routinely monitored by food inspection agencies) is co-injected along with a mixture of the C4-C18 APSA standards into a reversed phase liquid chromatography-mass spectrometry system. The system comprises an LC-ESI-QqQ (QTRAP 4000) instrument with an Agilent 2.7 m-Poroshell 120 SB-C18 column (2.1150 mm). Elution conditions comprise mobile phase: A=water, B=MeCN/water (95:5), both with 50 mM formic acid+5 mM ammonium hydroxide at pH 2.2; with a gradient of 5-100% B over 30 min at a hold time of 5 min, flow rate of 0.25 ml/min, and column temperature of 35 C. The liquid chromatography (LC) column provides separation of the complex mixture while the mass spectrometer provides detection of both the reference compounds and the co-injected APSA standards. The characteristic retention times of the reference compounds and the APSAs are measured. FIG. 6 depicts the LC-MS analysis of the mixture of reference compounds and co-injected APSA standards. The upper plots show the signals for drugs (not all the drug peaks are labeled) while the bottom plot shows the signals for the APSA standards that were co-injected with drugs.

(19) An interpolation of the retention times of the reference compounds into a fitted curve of the plot of retention time versus retention index value for the APSAs (FIG. 7) results in a measured retention index for the reference compounds. The curve fitting is done using the method of cubic splines. The resulting retention index of the reference compounds are then entered into a database, for example Table 2. Table 2 provides a listing of the reference compounds analyzed in FIG. 6 along with their measured retention times (RT) and calculated retention indices (RI). The standard deviation (SD) value shows the excellent reproducibility measured from 5 repeat analyses on the same day.

(20) TABLE-US-00004 TABLE 2 Name of Compound Abbrev SRM transition Avg. RT (min) Avg. RI SD (n = 5) RI Sulfonamides Sulfanilamide SNA 173.1 > 92.1 2.70 379.7 1.3 Sulfacetamide SAA 215.1 > 156.1 5.58 471.9 0.6 Sulfadiazine SDZ 251.1 > 156.1 6.42 500.1 0.4 Sulfathiazole STZ 256.1 > 156.1 7.29 530.4 0.4 Sulfapyridine SPR 250.1 > 156.1 7.34 531.9 0.2 Sulfamerazine SMR 265.1 > 92.1 7.75 546.8 0.4 Sulfamethazzine SMZ 279.1 > 186.1 8.76 584.0 0.3 Sulfamethizole SML 271.1 > 156.1 9.19 600.3 0.2 Sulfamethoxypyridazine SMP 281.1 > 126.1 9.25 602.9 0.2 Sulfachloropyridazine SCP 285.1 > 156.1 10.52 653.9 0.3 Sulfadoxine SDO 311.1 > 156.1 11.07 676.9 0.3 Sulfamethoxazole SMO 254.1 > 156.1 11.25 685.1 0.2 Sulfaquinoxaline SQO 301.1 > 156.1 13.24 780.4 0.2 Nitroimidazoles 1-(2-Hydroxyethyl)-2- MNZ-OH 188.1 > 123.1 3.95 419.5 0.5 hydroxymethyl-5- nitroimidazole 2-Hydroxymethyl-1- HMMNI 158.1 > 140.1 5.00 453.1 0.2 methyl-5-nitrimidazole Metronidazole MNZ 172.1 > 128.1 5.13 457.3 0.5 Dimetridazole DMZ 142.1 > 96.1 5.90 482.6 0.3 Ronidazole RNZ 201.1 > 55.1 6.22 493.3 0.4 1-Methyl-2-(2- IPZ-OH 186.1 > 168.1 8.93 590.6 0.2 hydroxyisopropyl)-5- nitroimidazole Ipronidazole IPZ 170.1 > 109.1 11.07 676.9 0.4 Fluoroquinolones Ciprofloxacin CIPRO 332.2 > 245.1 9.20 601.0 0.2 Danofloxacin DANO 358.2 > 96.1 9.54 614.1 0.3 Enrofloxacin ENRO 360.2 > 316.2 9.82 625.2 0.4 Sarafloxacin SARA 386.2 > 342.2 10.82 666.2 0.3 Quinolones Oxolinic acid OXA 262.1 > 216.1 12.53 744.6 0.6 Macrolides Erythromycin ERY 734.5 > 158.1 14.23 833.3 0.6 Dyes Leucocrystal Violet LCV 374.4 > 358.1 10.68 660.3 0.6 Leucomalachite green LMG 331.1 > 316.1 16.76 983.2 0.9 Malachite Green MG 329.2 > 313.1 19.84 1180.8 1.2 Crystal Violet CV 372.4 > 356.1 23.31 1406.5 1.1 Brilliant Green BG 385.3 > 341.1 25.90 1572.6 1.0

Example 3: Use of APSA Retention Index Standards to Predict Retention Times for Analytes in Order to Establish Windows for Scheduled SRM Analysis by LC-MS

(21) A database of retention indices for reference compounds (e.g., Table 2) is used to predict retention times of the same compounds that are to be run on columns with different dimensions, possibly with using various gradient conditions or different LC instruments. This can facilitate the establishment of retention windows of targeted analytes in order to permit the programming of a scheduled selected reaction monitoring method. The process involves first performing an analysis of a mixture of the APSA retention index standards under the LC conditions and on the column and instrument to be used for samples. Interpolation of the database retention indices for reference compounds into a plot of the retention times vs. retention index values for APSA standards would allow the calculation of retention times expected under those new conditions.

(22) FIG. 8 depicts a plot of the retention time vs. retention index values for APSA standards measured in three different LC-MS analyses using two different columns (150 vs 50 mm length) and two different gradients. A cubic spline fit has been generated to fit the data. Table 3 presents the prediction of drug retention times on a 502 mm column using the retention indices documented in in Table 2 and the plot shown in FIG. 8.

(23) TABLE-US-00005 TABLE 3 SRM Database Predicted Drug Class Name of Compound Abbrev transition RI RT (min) Sulfonamides Sulfanilamide SNA 173.1 > 92.1 379.7 0.8 Sulfacetamide SAA 215.1 > 156.1 471.9 2.3 Sulfadiazine SDZ 251.1 > 156.1 500.1 2.9 Sulfathiazole STZ 256.1 > 156.1 530.4 3.7 Sulfapyridine SPR 250.1 > 156.1 531.9 3.7 Sulfamerazine SMR 265.1 > 92.1 546.8 4.1 Sulfamethazzine SMZ 279.1 > 186.1 584.0 5.2 Sulfamethizole SML 271.1 > 156.1 600.3 5.6 Sulfamethoxypyridazine SMP 281.1 > 126.1 602.9 5.7 Sulfachloropyridazine SCP 285.1 > 156.1 653.9 7.1 Sulfadoxine SDO 311.1 > 156.1 676.9 7.6 Sulfamethoxazole SMO 254.1 > 156.1 685.1 7.8 Sulfaquinoxaline SQO 301.1 > 156.1 780.4 9.9 Nitroimidazoles 1-(2-Hydroxyethyl)-2- MNZ-OH 188.1 > 123.1 419.5 1.4 hydroxymethyl- 5-nitroimidazole 2-Hydroxymethyl-1- HMMNI 158.1 > 140.1 453.1 2.0 methyl-5-nitrimidazole Metronidazole MNZ 172.1 > 128.1 457.3 2.0 Dimetridazole DMZ 142.1 > 96.1 482.6 2.5 Ronidazole RNZ 201.1 > 55.1 493.3 2.8 1-Methyl-2-(2- IPZ-OH 186.1 > 168.1 590.6 5.4 hydroxyisopropyl)-5- nitroimidazole Ipronidazole IPZ 170.1 > 109.1 676.9 7.6 Fluoroquinolones Ciprofloxacin CIPRO 332.2 > 245.1 601.0 5.7 Danofloxacin DANO 358.2 > 96.1 614.1 6.0 Enrofloxacin ENRO 360.2 > 316.2 625.2 6.3 Sarafloxacin SARA 386.2 > 342.2 666.2 7.4 Quinolones Oxolinic acid OXA 262.1 > 216.1 744.6 9.2 Macrolides Erythromycin ERY 734.5 > 158.1 833.3 10.9 Dyes Leucocrystal Violet LCV 374.4 > 358.1 660.3 7.2 Leucomalachite green LMG 331.1 > 316.1 983.2 13.5 Malachite Green MG 329.2 > 313.1 1180.8 16.3 Crystal Violet CV 372.4 > 356.1 1406.5 19.3 Brilliant Green BG 385.3 > 341.1 1572.6 21.5

(24) Other advantages that are inherent to the invention are obvious to one skilled in the art. The embodiments are described herein illustratively and are not meant to limit the scope of the invention as claimed. Variations of the foregoing embodiments will be evident to a person of ordinary skill and are intended by the inventor to be encompassed by the following claims.