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Ultrahigh-performance liquid chromatography (UHPLC) has brought two important innovations to LC-MS analysis. First is the use of 1.8-µm columns, which provides an increase in plate number from the 5-µm columns of about a factor of two.This is important for environmental applications where complex matrices are encountered, such as pharmaceuticals in wastewater analysis. However, the use of 1.8-µm columns requires higher-pressure pumps operating at pressures that exceed 600 bar on a routine basis, which is the range commonly used for UHPLC. Secondly, the 1.8-µm column allows the use of Rapid Resolution Liquid Chromatography (RRLC) (Agilent Technologies, Santa Clara,CA) and even ultrafast chromatography, where runs may be less than 1 min in length. The high flow rates required for these analyses create pressures on the order of 800–1000 bar, or more, and are clearly in the range of UHPLC.
The demands of high sample throughput in short time frames have given rise to high efficiency and fast liquid chromatography using the 1.8-µm reversed-phase columns. Fast chromatography has become a necessity in laboratories that analyze hundreds of samples per day or those needing short turnaround times. Using RRLC, results of a sample batch can be reported in a few hours rather than a few days. In both the water quality and food industries, regulatory laboratories produce validated results in less than an hour so that water treatment may proceed or vegetable shipments can be released the same day they are measured or produced. The end result is greater productivity for users and greater cost efficiency for the reporting laboratory. Thus, productivity is improved by shortened analysis time, which typically requires UHPLC. The definition of Rapid Resolution Liquid Chromatography is simple. Liquid chromatographic separations that are less than 10 min are fast, and separations less than 1 min are widely known as ultrafast.1
The other aspect of UHPLC is the increased peak capacity available when longer columns with 1.8-µm packing are used. It is now possible to have almost 300 times greater peak capacity, which is a valuable asset to unknown analysis in wastewater and other environmental applications such as pesticide screening. Finally, the UHPLC system should be robust and capable of both high pressure and high flow (>1 mL/min at pressures up to 1200 bar) to carry out both Rapid Resolution and normal flow chromatography with high peak capacity. Specially designed for pressures to 1200 bar (18,000 psi), 1.8-µm columns from Agilent Technologies give a variety of phases (C8, C18 in both Stable Bond and ZORBAX Eclipse Plus formats). These are useful for difficult water samples, as this article will show, including improved peak capacity for U.S. EPA Method 1694 for pharmaceuticals in wastewater. They are also useful for rapid resolution of pharmaceuticals and pesticides using both triple quadrupole mass spectrometry as well as liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS).
Experimental
The work shown here was carried out by Drs. Imma Ferrer and Michael Thurman at the Center for Environmental Mass Spectrometry at the University of Colorado, Boulder, using the Agilent 1290 Infinity LC system and both the Agilent 6430 triple quadrupole LC-MS and Agilent 6220 accurate-mass time-of-flight LC-MS system.
Columns
Two different columns were tested for Rapid Resolution High-Throughput (RRHT) analyses, including the high-pressure (1000 bar) 1.8-µm particle sizes. Table 1 lists the columns tested in this work and their theoretical plates.
Chromatographic and mass spectral conditions
The Agilent 1290 Infinity LC was used for all UHPLC chromatographic separations, and the Agilent 1200 Series SL was used for the standard U.S. EPA Method 1694. The conditions were as follows for each figure.
Figure 1 - Reduction of analysis time from 30 min to 10 min using the
Agilent 1290 Infinity with UHPLC for Group 1 pharmaceuticals in U.S. EPA
Method 1694.
In Figure 1a, the liquid chromatograph was the Agilent 1200 Series SL. The gradient was from 10% acetonitrile/water to 100% acetonitrile in 30 min with a 5-min hold time. The flow rate was 0.6 mL/min. The column was the ZORBAX Eclipse Plus-C18, 4.6 mm × 150 mm, 3.5 µm. Peak widths at the base were 15–18 sec with peak capacity of 100. Maximum pressure was 75 bar.
The mass spectrometer was the Agilent 6410 triple quadrupole LC-MS system in electrospray positive mode with three time segments in multiple reaction monitoring (MRM) mode. There were two transitions per compound with 10-msec dwell time for each transition. The compounds were Group 1 of U.S. EPA Method 1694. (See Ref. 2 for further detail on compounds and their transitions.)
In Figure 1b, the liquid chromatograph was the Agilent 1290 Infinity LC. The gradient was from 10% acetonitrile/water to 100% acetonitrile in 10 min with a 1-min hold time. The flow rate was 0.6 mL/min. The column was the ZORBAX Eclipse Plus-C18, 2.1 mm × 50 mm, 1.8 µm. Peak widths at the base were 5–6 sec with peak capacity of 100. Maximum pressure was 375 bar.
Figure 2 - Increased peak capacity showing the separation of the entire list of U.S. EPA Method 1694 pharmaceuticalsplus 15 new compounds for a total of 90 pharmaceuticals in less than 20 min using a ZORBAX Eclipse Plus-C18, 2.1 mm × 100 mm, 1.8-µm packing material with UHPLC using the Agilent 1290 Infinity LC. Peaks were 5–6 sec wide and peak capacity was 200.
The mass spectrometer was the Agilent 6430 triple quadrupole LC-MS in electrospray positive mode with one time segment in MRM mode. There were two transitions per compound with 10-msec dwell time for each transition. The compounds were Group 1 of U.S. EPA Method 1694. (See Ref. 2 for further detail on compounds and their transitions.)
In Figure 2, the liquid chromatograph was the Agilent 1290 Infinity LC. The gradient was from 10% acetonitrile/water to 100% acetonitrile in 20 min with a 2-min hold time. The flow rate was 0.6 mL/min. The column was the ZORBAX Eclipse Plus-C18, 2.1 mm × 100 mm, 1.8 µm. Peak widths at the base were 5–6 sec with peak capacity of 200. Maximum pressure was 750 bar.
The mass spectrometer was the Agilent 6430 triple quadrupole LC-MS in electrospray positive mode with one time segment in MRM mode. There was one transition per compound with 10-msec dwell time for each transition. The compounds were Groups 1–4 of U.S. EPA Method 1694, plus 15 additional pharmaceuticals. (See Ref. 2 for further detail on compounds and their transitions.)
Figure 3 - a) Ultrafast gradient for 12 pharmaceutical standards. b)
Ultrafast gradient for wastewater from Boulder, CO. The compounds
detected included carbamazepine, continine, caffeine, diphenhydramine,
thiabendazole, and trimethoprim.
In Figure 3a, the liquid chromatograph was the Agilent 1290 Infinity LC. The gradient was from 10% acetonitrile/water to 100% acetonitrile in 2 min without a hold time. The flow rate was 1.2 mL/min. The column was the ZORBAX Eclipse Plus-C18, 2.1 mm × 50 mm, 1.8 µm. Peak widths at the base were 1–3 sec with peak capacity of 60. Maximum pressure was 780 bar.
The mass spectrometer was the Agilent 6430 triple quadrupole LC-MS in electrospray positive mode with two time segments in MRM mode and six compounds per segment. There was one transition per compound with a 5-msec dwell time for each transition. The compounds were a selected set of 12 compounds from U.S EPA Method 1694.
In Figure 3b, the liquid chromatograph was the Agilent 1290 Infinity LC. The gradient was from 10% acetonitrile/water to 100% acetonitrile in 2 min without a hold time.The flow rate was 1.2 mL/min. The column was the ZORBAX Eclipse Plus-C18, 2.1 mm × 50 mm, 1.8 µm. Peak widths at the base were 1–3 sec with peak capacity of 60. Maximum pressure was 780 bar.
The mass spectrometer was the Agilent 6430 triple quadrupole LC-MS in electrospray positive mode with two time segments in MRM mode and three compounds per segment. There was one transition per compound with a 5-msec dwell time for each transition. The compounds were carbamazepine, continine, caffeine, diphenhydramine, thiabendazole, and trimethoprim from U.S. EPA Method 1694.
Figure 4 - Pesticide analysis of over 100 compounds by LC/TOF-MS in less than 2 min.
Figure
5 - Peak width of 0.7 sec at half-height using the Agilent 1290
Infinity with a ZORBAX Eclipse Plus-C18, 2.1 mm × 50 mm with a flow rate
of 1.5 mL/min at a pressure of 900 bar.
In Figures 4 and 5, the liquid chromatograph was the Agilent 1290 Infinity LC. The gradient was from 10% acetonitrile/water to 100% acetonitrile in 2 min without a hold time. The flow rate was 1.5 mL/min. The column was the ZORBAX Eclipse Plus-C18, 2.1 mm × 50 mm, 1.8 µm. Peak widths at the base were 1–3 sec with peak capacity of 60. Maximum pressure was 900 bar.
The mass spectrometer was the Agilent 6430 triple quadrupole LC-MS in electrospray positive mode in 2-GHz mode with 20 scans per sec at a mass accuracy of >2 ppm. The compounds included a mix of 220 pesticides from the list reported in Ref. 3.
Figure 6 - Effect of dwell time and data points per peak and how they affect the peak shape for a single compound, carbamazepine, from a 1-sec peak at >20 cycles per sec (cps) to 3–4 sec peak at 3 cycles per sec (cps).
In Figure 6, the liquid chromatograph was the Agilent 1290 Infinity LC. The gradient was from 10% acetonitrile/water to 100% acetonitrile in 2 min without a hold time. The flow rate was 1.2 mL/min. The column was the ZORBAX Eclipse Plus-C18, 2.1 mm × 50 mm, 1.8 µm. Peak widths at the base varied as a function of scans per second on the mass spectrometer. Maximum pressure was 780 bar.
The mass spectrometer was the Agilent 6430 triple quadrupole LC-MS in electrospray positive mode with one time segment in MRM mode and one compound per segment, carbamazepine. There was one transition per compound with dwell times that varied from 1 to 300 msec and were equal to a range of 3 to greater than 20 scans per sec.
Figure 7 - Effect of retention time on S/N and the area counts for caffeine in LC-MS analysis.
In Figure 7, the liquid chromatograph was the Agilent 1290 Infinity LC. The gradient was from 10% acetonitrile/water to 100% acetonitrile in 2 min without a hold time, 6 min without a hold time, and 30 min without a hold time. The flow rate was 0.6 mL/min. The column was the ZORBAX Eclipse Plus-C18, 2.1 mm × 100 mm, 1.8 µm. Peak widths at the base were 2–6 sec with peak capacity of 60. Maximum pressure was 750 bar.
The mass spectrometer was the Agilent 6430 triple quadrupole LC-MS in electrospray positive mode with one time segment in MRM mode and one compound per segment, caffeine. There was one transition per compound with a dwell time of 5 msec.
Sample preparation
Pharmaceutical analytical standards were purchased from Sigma (St. Louis, MO). Individual pharmaceutical stock solutions (approximately 1000 g/mL) were prepared in pure acetonitrile or methanol, depending on the solubility of each individual compound, and stored at –18 ºC. From these solutions, working standard solutions were prepared by dilution with acetonitrile and water.
Pesticide analytical standards were purchased from Dr. Ehrenstorfer GmbH (Ausburg, Germany). Individual pesticide stock solutions (1000 g/mL) were prepared in pure acetonitrile or methanol, depending on the solubility of each individual compound, and stored at –18 ºC. From these concentrated solutions, working standard solutions were prepared by dilution with acetonitrile and water.
Wastewater samples were collected from an effluent site in Boulder Creek, CO, and extracted with polymeric cartridges using a modified U.S. EPA protocol. One-liter water samples were extracted directly onto a 500-mg cartridge without pH adjustment, dried for 10 min with air, and eluted with 8 mL of methanol. The methanol was evaporated to 1 mL and analyzed by LC-MS-MS as described below. “Blank” wastewater extracts were used to prepare the matrix matched standards for validation purposes. The wastewater extracts were spiked with the mix of pharmaceuticals at different concentrations (ranging from 0.1 to 500 ppb), and then analyzed by LC-MS-MS.
Results and discussion
This article contains three sections discussing examples of peak capacity and Rapid Resolution for pharmaceuticals in wastewater using U.S. EPA Method 1694, UHPLC, and LC/TOF-MS of pesticides, and some important chromatographic considerations with UHPLC-MS.
Environmental pharmaceutical analysis by LC-MS-MS
U.S. EPA Method 1694 is a standard method requiring 20-min analysis times or longer to satisfy the method requirements. However, recent changes published by the U.S. EPA suggest that other chromatographic conditions may be used, such as shorter analysis times and Rapid Resolution, if sufficient mass spectrometric analysis is used (for example, two transitions per compound). Figure 1 shows the use of the Agilent 1290 Infinity with UHPLC to reduce analysis times from 30 min to 10 min, a 66% decrease in analysis times with a peak capacity of 100 in both analyses. The original U.S. EPA method called for a ZORBAX Eclipse Plus-C18, 3.5-µm column, and the Rapid Resolution is with the ZORBAX Eclipse Plus-C18 2.1 mm × 50 mm, 1.8-µm column.
Because the pressure is at 375 bar, it is possible to easily increase peak capacity and the number of pharmaceuticals that may be separated by substituting a longer column (2.1 mm × 100 mm) and maintaining the same flow rate of 0.6 mL/min. This doubles the pressure from 375 to 750 bar. The results are shown in Figure 2.
It is also possible to perform ultrafast chromatography with the Agilent 1290 Infinity LC for pharmaceuticals using a triple quadrupole LC-MS. In the example shown in Figure 3, the number of compounds has been reduced to 12 in order to obtain at least 20 scans across each peak. By obtaining 20 scans or more, the peak width may be reduced to 1–2 sec, and the result is ultrafast chromatography. The column is a ZORBAX Eclipse Plus-C18, 2.1 mm × 50 mm, with a flow rate of 1.2 mL/min and a 1.5-min gradient. The 12 pharmaceuticals eluted in less than 60 sec. This, to the authors’ knowledge, is the first example of ultrafast chromatography applied to an environmental sample. In this case, it is a wastewater from Boulder, CO, that contains the following pharmaceuticals: carbamazepine, continine, caffeine, diphenhydramine, thiabendazole, and trimethoprim.
It is important to realize that when using ultrafast gradient conditions, quality assurance and quality control data are required. Therefore, all sample purification measures must be taken during sample preparation to minimize suppression. It is also necessary to use labeled internal standards for quantitation since the entire sample matrix is eluting in a very narrow window. The reproducibility of the 1290 Infinity was within 1 sec, making it easy to obtain reliable data. Also important is the use of at least two transitions by LC-MS-MS, one for quantitation and the other as a qualifier ion. (See Ref. 2 for further examples.)
UHPLC and LC-TOF-MS
One of the major concerns when doing RRLC is obtaining good sampling across the narrow peaks of 1–2 sec using mass spectrometry. The previous examples showed how this is done using triple quadrupole LC-MS and maintaining 20 scans across each peak. The use of LC/TOF-MS and LC/Q-TOF-MS makes this task easy for as many compounds as one would like to monitor. This is because the TOF-MS instruments obtain data in full spectrum mode at all times. It is merely necessary to set the software to obtain the 20 spectra per sec for the TOF-MS instruments. Figure 4 shows how effective this strategy is when performing UHPLC with either LC/TOF-MS or LC/QTOF-MS. More than 100 pesticides were analyzed in less than 80 sec using the Agilent 1290 Infinity LC with a ZORBAX Eclipse Plus-C18, 2.1 mm × 50 mm column at a flow rate of 1.5mL/min. The UHPLC was required as pressures reached 900 bar. The separation included peak widths at half-height of only 0.7 sec. See Figure 5 for the herbicide, terbutryn.
The chromatographic speed, separation, and power of LC/TOF-MS make it possible to analyze complex mixtures of pesticides from food or water matrices in a few minutes. This allows the rapid analysis used in the food monitoring industry in Europe, where shipments of vegetables and fruits are not unloaded until the analysis of pesticides is complete. Therefore, rapid analysis is important in these cases.
Chromatographic considerations with UHPLC-MS
Optimization of chromatographic and mass spectrometry conditions for the best use of UHPLC includes the following ideas. First, it is important to have at least 20 MS data points across each peak in order to obtain the 1–2 sec peak widths of ultrafast chromatography. An illustration of the importance of data cycles per second is shown in Figure 6.
The peak broadening that appears at the lower cycle rates is caused by a smoothing routine meant to shape peaks for good integration and quantitation, and is a common procedure in all chromatographic software. Therefore, when using triple quadrupole LC-MS instruments, it is recommended to use a short dwell time of 5 msec and the dynamic MRM procedures to ensure that 20 cycles per peak are obtained. In the case of LC/TOF-MS and LC/Q-TOF-MS instruments, it is only necessary to set the software to obtain 20 spectra per sec across the mass range that is acquired.
Secondly, quantitative aspects of analysis are a consideration in good UHPLC practice. Here, it is important that maximum peak sensitivity be obtained. Figure 7 shows an example where caffeine is maximized for peak intensity and peak area by adjusting the gradient and flow rate until the maximum signal is obtained. In this case, a 6-min gradient resulted in the optimum signal-to-noise (S/N) ratio of 180 and an area count of 55,000 counts at a retention time of 1.7 min. Note that the signal-to-noise drops to half at a value of 91 with the longer gradient of 30 min, but a retention time increase of only 0.2 min. Thus, it is important to test various flow rates and retention times to optimize signal strength, especially of the polar and early eluting compounds in a chromatographic analysis.
A final consideration in good UHPLC is the suppression of LC-MS signal. It was mentioned earlier and must be emphasized that standards in fast and ultrafast analysis will often show little or no suppression because of the purity of the standard mixture. However, real samples may show suppression; therefore, it is important to dilute samples or to purify them in extraction procedures to limit the amount of matrix that is present. Finally, it is valuable to use deuterated or C-13 labeled standards when measuring pharmaceuticals in wastewater and other complex matrices. This is the recommended procedure of U.S. EPA Method 1694.2
Conclusion
ZORBAX Eclipse Plus-C18, 2.1 mm × 50 mm columns are recommended for Rapid Resolution and ultrafast chromatographic separations, while for maximum peak capacity the ZORBAX Eclipse Plus-C18, 2.1 mm × 100 mm is a better choice. The authors’ results show that fast flow rates greater than 1.5 mL/min may be used, and pressures greater than 1000 bar are possible with confidence and reliability. Finally, the Agilent 1290 Infinity LC is a good example of the evolution of chromatography from the gravity columns of Tswett to the UHPLC realm of ultrafast high pressure liquid chromatography made easy.
Please check out our Ultrahigh-performance liquid chromatography (UHPLC) section for more information or to find manufacturers that sell
these products.
References
- U.S. EPA Method 1694: Pharmaceuticals and personal care products in water, soil, sediment, and biosolids by HPLC/MS/MS, Dec 2007, EPA-821-R-08-002.
- Ferrer, I.; Thurman, E.M.; Zweigenbaum, J.A. EPA Method 1694: Agilent’s 6410A LC/MS/MS Solution for Pharmaceuticals and Personal Care Products in Water, Soil, Sediment, and Biosolids by HPLC/MS/MS, 2008, Application Technologies publ. 5989-9665EN.
- Thurman, E.M.; Ferrer, I.; Zweigenbaum, J.A. Multi-Residue Analysis of 301 Pesticides in Food Samples by LC/Triple Quadrupole Mass Spectrometry, 2008, Application Technologies publ. 5989-6414.
The authors are with the Center for Environmental Mass Spectrometry, Department of Environmental Engineering, University of Colorado, 1111 Engineering Dr., 422 UCB, Boulder, CO 80309-0422, U.S.A.; tel.: 303-492-5071; fax: 303-492-2199; e-mail: [email protected]. The Center for Environmental Mass Spectrometry acknowledges the help and advice of Drs. Jerry Zweigenbaum, Michael Woodman, and Peter Stone of Agilent Technologies, Inc. (Wilmington, DE).