An Improved Method for HPLC Determination of Acidic Herbicides in Aqueous Samples

Transcription

1 Volume VI Issue 5, 1997 An Improved Method for HPLC Determination of Acidic Herbicides in Aqueous Samples Michael S. Young, Waters Corporation solvent suitable for multiple analyses. Moreover, such an extract is generally much more stable than the aqueous sample from which it was derived, and is therefore more suitable for long-term storage. Also, the off-line approach allows the processing of many samples at one time, an approach which is generally more productive in laboratories that are not fully automated. Table of Contents Acidic Herbicides Faster Gradient Chromatography Introduction The phenoxyacetic acid and other acidic herbicides are among the most widely used agrochemicals. Most of these compounds are typically determined in water samples following liquid-liquid extraction procedures or by reversed-phase solid-phase extraction (SPE). The Environmental Protection Agency (EPA) in the United States currently offers two SPE procedures for the analysis of drinking water for these compounds. One of these, Method 555 [1], is an on-line SPE procedure followed by HPLC analysis. The sample is first adjusted to ph 12 to hydrolyse esterified analytes, then it is acidified to ph 1 and a 20 ml aliquot is pumped through a reversed-phase concentrator column. By use of a switching valve, the concentrator column is then plumbed in line with the analytical column and the sample constituents are then passed to the analytical column for separation and detection. While this method gives acceptable performance, there are some important limitations which are dealt with in this study. Although an on-line procedure is convenient for some analysts, many prefer the off-line approach which gives a convenient extract in an organic Simple and Fast Methods Development for HPLC and Sample Preparation What s ew How to Reach Us Application otes Trazodone Sulfonylurea Herbicides in Groundwater Editor: Uwe D. eue, Ph. D. ISS #

2 Recently, a new type of reversed-phase analytical HPLC column was introduced [2]. The packing in this column is SymmetryShield RP 8 5 µm, prepared from Symmetry silica by bonding to it an alkyl chain containing an imbedded polar carbamate functionality. This results in a packing material on which polar functionalities on the silica surface are effectively shielded from interactions with polar analytes. Outstanding peak symmetry and unique selectivity are therefore shown for many types of analytes which often tail or may show insufficient resolution on traditional reversed-phase columns. Among the classes of compounds for which SymmetryShield RP 8 is particularly useful is the acidic herbicides. This new HPLC column allows the separation of these analytes in one analytical run where previously recommended columns required two runs. Another new product is the Oasis HLB sample extraction cartridge for reversedphase solid-phase extraction (SPE). These cartridges contain a water-wettable, moderately polar polymeric resin of high sorbent capacity. The high capacity allows the analysis of samples as large as 250 ml using a cartridge containing 60 mg of resin. Consequently, when the cartridge is eluted with 1 ml of solvent, considerable sample enrichment is achieved in a 15 minute procedure. Also, because the adsorbed sample is eluted with relatively small solvent volumes of about 1 ml, subsequent exchange to HPLC mobile phase is rapid and straightforward. Because the polymer is water wettable, the analyst need not worry if the cartridge bed runs dry during the analysis or during any of the conditioning steps. This cartridge has recently been demonstrated to be highly effective for the extraction of a wide variety of polar herbicides and metabolites from drinking water samples [3]. The first goal of this work was to develop an improved acidic herbicides sample preparation procedure that would be compatible with either GC or LC. The other goal was to develop an improved HPLC analysis. We investigated an alternative EPA protocol for acidic herbicides as the basis of an improved method that would meet both objectives. The alternative EPA method for determination of acidic herbicides is method which utilises off-line SPE followed by derivatisation and GC analysis [4]. With some modifications, a sample preparation procedure similar to that used in the EPA GC method was successfully applied to the development of a new HPLC based method. This new HPLC method incorporates the Oasis HLB cartridge for sample preparation and the SymmetryShield RP 8 column for the subsequent HPLC analysis. Experimental Instrumentation and Materials HPLC System. The Waters liquid chromatography system used for this study was comprised of a 616 Solvent Delivery System, a 996 Photodiode Array Detector (PDA), and a 712 Autosampler. Data processing and instrument control were accomplished using the Millennium Chromatography Manager. Temperature was ambient. The primary column used for these experiments was a SymmetryShield RP 8 5 µm, 3.9 mm I.D. x 150 mm. Reagents. Reagents and solvents for preparation of mobile phases were obtained from Baker (Phillipsburg, J). HPLC grade water was obtained inhouse using a Milli-Q system (Millipore, Bedford, MA). Standard Chemicals. Chemical standards for calibration and for spiking experiments were obtained from Accustandard (ew Haven, CT). Structures for the herbicides included in this study are presented in Figure 1. Water Samples. Two types of water were used for spiking experiments. Municipal finished drinking water (tap water) was obtained from the laboratory faucet at Waters Corporation in Milford, MA. Groundwater was obtained from a well at the home of the author in Stow, MA. Solid-Phase Extraction. All SPE analysis was performed using Waters Oasis HLB extraction cartridges (3 cc/60 mg sorbent). Procedure Sample Preparation. The samples (75 ml) were first spiked with the appropriate amount of the analytes and were then adjusted to ph 12 with aoh. After 1 hour, the samples were adjusted to ph 2 with phosphoric acid. A vacuum manifold was fitted with 60 ml reservoirs (75 ml total capacity) atop Oasis HLB 3 cc cartridges. An aspirator was used to provide a vacuum of 30 to 50 kpa below atmospheric pressure to yield a flow of about 5 ml/minute through the cartridges for conditioning and sample loading steps. Before the samples were loaded, the cartridges were conditioned with 3 ml of 10% methanol (MeOH) in methyl t-butyl ether (MTBE), then 3 ml of methanol, and finally 2 ml of ph 2 reagent water. After sample loading was complete, the cartridges were washed with 1 ml of reagent water and then eluted with 2 ml of 10% methanol in MTBE. Elution was accomplished at a very low vacuum level to give a flow of about 2 ml/minute. The eluted samples were then evaporated under a gentle stream of nitrogen (37 C) to approximately 100 µl and then adjusted to exactly 500 µl with water. o stopcocks were employed in manipulation of flow rate during cartridge conditioning steps or in sample loading. o effort was made to prevent the sorbent bed from drying. HPLC Analysis. The following conditions were used for the separation with the SymmetryShield RP 8 column. The aqueous portion of the mobile phase (mobile phase A) was 13 mm sodium phosphate buffer at ph 3.4. The organic portion of the mobile phase was acetonitrile. A multistep gradient was employed to separate all analytes included in this study. The gradient was 85% A initial with a linear gradient to 70% A in 8 minutes, held at 70% A until 15 minutes, then linear to 40% A in 30 minutes, and then to 10% A in 35 minutes. The flow rate was 1.0 ml/min and the injection volume was 75 µl. 2

3 Results The solvent chosen for the elution of the Oasis HLB cartridges was 10% methanol in methyl t-butyl ether (10% MeOH/MTBE), the solvent specified for the EPA GC analysis method. This solvent is used for the GC method because it gives good recovery and is compatible with the diazomethane derivatisation required for the analysis. In this study, this mixed solvent was shown to have some important attributes useful for an HPLC procedure. A significant interference is usually present in many drinking water samples extracted by reversed-phase (SPE) solidphase extraction at acidic ph. This interference is caused by the presence of humic or other natural organic matter (OM). This interference is most pronounced when high polarity solvents, such as methanol or tetrahydrofuran (THF), are used for SPE elution; this interference is minimised if relatively non-polar solvents are employed. Dichloromethane and MTBE were both investigated as potential elution solvents, but recoveries were generally unacceptably low. However, 10% MeOH/MTBE gave recovery in the same range as THF and was sufficiently non-polar to minimise the interference of natural organic matter (OM). Care was taken to remove the MTBE from the eluent prior to HPLC analysis; this was effectively accomplished by evaporation of the eluent to 100 µl and subsequent dilution with water. The results from the spiked water experiments are presented in Table 1. They compare favorably to those reported in both EPA methods, with the exception of dinoseb. In Table 1, results for the first three experiments were obtained by evaporating the eluent to dryness before reconstituting in 15% methanol/water. It was observed that dinoseb was not consistently recovered using that procedure. The high and consistent recovery seen in the last experiment was obtained by evaporative concentration of the eluent to 100 µl and dilution to 500 µl with water. Figure 1: Structures of herbicides included in this study Picloram Dicamba Chloramben 4-itrophenol Bentazon 2,4-D MCPA Dichlorprop H 2 H 2 COOH HOOC OCH 3 OH O 2 O SO 2 H COOH OCH 2 COOH OCH 2 COOH CH 3 CH 3 OCHCOOH 2,4,5-T MCPP 3,5-Dichlorobenzoic acid 2,4-DB 2,4,5-TP Acifluorfen Dinoseb F 3 C O 2 OCH 2 COOH CH 3 OCHCOOH CH 3 COOH O(CH 2 ) 3 COOH O OH O 2 CH 3 OCHCOOH COOH O 2 3

4 The HPLC analysis is shown in Figure 2. This chromatogram was obtained from the 400 ng/l spiked tap water experiment and shows the excellent performance obtained for this separation using the SymmetryShield RP 8 column. Although other HPLC columns are routinely used for this analysis, it is difficult to obtain the separation of all 15 of these compounds on other phases in a single run. Conclusions A convenient, sensitive, off-line SPEbased method has been developed for the determination of acidic herbicides in aqueous samples. This method is compatible with either HPLC or GC analysis. Of critical importance to the HPLC analysis procedure is the outstanding performance of the Waters SymmetryShield RP 8 column for the separation of the acid herbicides. Using Oasis HLB cartridges and the improved HPLC analysis developed in this study, analysis time is significantly reduced compared with the derivatisation GC method. Unlike the on-line SPE/HPLC method, the new procedure discussed in this study yields a convenient extract which can be analysed by multiple, confirmatory procedures, saved for later re-analysis, or held for long term storage. Table 1: Results of acidic herbicides recovery experiments, % recovery (RSD) Column: SymmetryShield RP 8, 5 µm, 3.9 mm x 150 mm Mobile phase: A: ph 3.4 phosphate buffer (13 mm) B: Acetonitrile Gradient: 85% A linear to 70% A in 8 min, hold until 15 min, then linear to 40% A in 30 min, then linear to 10% A in 35 min Flow rate: 1.0 ml/min Detection: UV at 230 nm (0.015 AUFS) Injection: 75 µl 1 Tap Water Tap Water Well Water Well Water 2.0 µg/l 400 ng/l 2.0 µg/l 400 ng/l 5 replicates 5 replicates 5 replicates 5 replicates Compound picloram 90.9 (7.0) 126 (5.3) 97.5 (3.8) 106 (2.3) dicamba 85.1 (7.2) 115 (4.4) 98.5 (3.8) 96.3 (8.3) chloramben 86.7 (7.3) 99.2 (6.9) 95.1 (10) 90.6 (5.6) 4-nitrophenol 83.3 (6.1) 113 (6.0) 90.4 (1.7) 112 (13) bentazon 89.3 (6.0) 114 (5.6) 91.2 (3.0) 104 (8.8) 2,4-D 92.3 (7.1) 107 (3.1) 86.5 (1.8) 122 (12) MCPA 97.6 (8.2) 104 (4.5) 80.8 (3.6) 96.7 (5.5) dichlorprop 96.4 (11) 107 (9.0) 87.4 (3.0) 103 (6.0) 2,4,5-T 106 (6.2) 116 (8.8) 95.1 (5.0) 96.6 (12) MCPP 100 (7.7) 116 (6.6) 93.8 (3.0) 94.7 (2.9) 3,5-dichlorobenzoic 93.3 (6.3) 119 (9.7) 84.3 (2.7) 96.9 (5.9) 2,4-DB 95.4 (5.1) 110 (8.4) 83.7 (5.6) 83.3 (5.2) 2,4,5-TP 89.3 (7.9) 92.5 (6.7) 87.7 (5.3) 82.7 (10) acifluorfen 94.8 (8.3) 102 (8.5) 70.0 (17) 81.3 (8.2) dinoseb 71.7 (7.1) 73.8 (6.8) 54.7 (5.2) 88.1 (1.9) Figure 2: HPLC analysis of 400 ng/l spiked water sample on a SymmetryShield RP 8 column 1. picloram 2. dicamba 3. chloramben 4. 4-nitrophenol 5. bentazon 6. 2,4-D 7. MCPA 8. dichlorprop 9. 2,4,5-T 10. MCPP 11. 3,5-dichlorobenzoic acid 12. 2,4-DB 13. 2,4,5-TP 14. acifluorfen 15. dinoseb 2 3 spiked tap water nonspiked sample Minutes 30 4

5 References 1. Methods for the Determination of Organic Compounds in Finished Drinking Water, Supplement 2 (1992), EPA/600/R-92129, p O Gara, J.E.; Alden, B.A.; Walter, T.H.; Peterson, J.S.; iederlander, C.L. and eue, U.D. Anal. Chem , p Young, M.S., HPLC 97, paper 177 Birmingham, England, June Methods for the Determination of Organic Compounds in Finished Drinking Water, Supplement 2 (1992), EPA/600/R-92129, p 51 Ordering Information SymmetryShield RP 8 5 µm Columns Dimension Part o. 2.1 x 150 mm WAT x 150 mm WAT x 150 mm WAT x 150 mm WAT x 250 mm WAT SymmetryShield RP 8 5 µm Cartridge Columns* Dimension Part o. 3.9 x 50 mm WAT x 150 mm WAT x 150 mm WAT x 250 mm WAT SymmetryShield RP µm Columns Dimension Part o. 2.1 x 30 mm WAT x 50 mm WAT x 100 mm WAT x 150 mm WAT x 50 mm WAT x 75 mm WAT x 100 mm WAT x 150 mm WAT SymmetryShield RP µm Cartridge Columns* Dimension Part o. 4.6 x 75 mm WAT x 100 mm WAT For information on the complete line of Symmetry columns, please check box 3 on the business reply card. Description Endfittings Integrated Guard Holder (for Waters steel cartridge columns only) Universal Guard Holder (for any HPLC column) Sentry SymmetryShield Guard Column** Part o. WAT WAT WAT WAT * Requires reusable endfittings ** Guard columns require the appropriate Sentry Guard Holder. (3.9 x 50 mm cartridge columns must use the Universal Guard Holder) For your free copy of the Symmetry Applications otebook, please check box 4 on the business reply card. 5

6 Achieve Faster Gradient Chromatography by Changing Column Dimensions Jeanne B. Li, Waters Corporation Introduction In today s world, everyone is trying to produce more results in the same amount of time. Analysts are often reluctant to make radical changes in the way they perform gradient chromatography or they do not have time to explore new columns or ideas. What has worked in the past is the safest way to proceed with methods development. This means chromatographers tend to continue using the same gradient shapes, run times, column lengths and diameters. However, over the years, there have been many improvements in column packing materials and techniques to provide more separation power, higher efficiencies and better column-to-column reproducibility. ow there are many different column diameters and lengths besides the commonly used 3.9 x 150 mm and 4.6 x 250 mm standard analytical columns. Using an HPLC system with a low system or gradient delay volume, one can take advantage of these newer column sizes to obtain faster gradients. This article presents the results of experiments on one family of columns, the Waters Symmetry C 18 columns. The separation was accomplished with a simple reversed-phase water-methanol gradient of acetone (V o marker) and the alkylphenones from C 2 to C 8. This homologous series of standards was chosen because they elute at evenly spaced intervals on a linear gradient. The goal of the experiments was to develop a chromatographic method with the shortest run time possible and still achieve baseline separation. Two strategies were used: 1) shorter columns and 2) higher flow rates. Experimental Conditions All experiments were performed on a Waters Alliance System, consisting of the Waters 2690XE Separations Module, a 996 Photodiode Array Detector and a Millennium 2010 Chromatography Manager. The outlet tubing from the injector was reduced to twelve inches in length. A minimum tubing length was used from the column to the detector: inch i.d. tubing for 3.9 mm i.d. columns or inch i.d. tubing for 2.1 mm i.d. columns. The measured system or gradient delay volume was 600 µl. Symmetry C 18 columns with internal diameters (i.d.) of 3.9 or 2.1 mm and lengths of 20, 30, 50 or 150 mm (Waters Corp., Milford, MA) were used for this study. The 3.9 mm columns contained 5 µm packing material. The 2.1 mm columns contained 3.5 µm material. The mobile phases consisted of HPLC-grade water (Milli-Q Plus, Millipore, Bedford, MA) and methanol (J.T. Baker, Phillipsburg, J). The analytes were acetone (J.T. Baker, Phillipsburg, J) and the alkylphenones (Aldrich Chemical Co., Milwaukee, WI). The eluates were monitored at 254 nm. A 30 minute linear gradient from 40 to 100% methanol was employed. The flow rates were 1.0 ml/min and 0.29 ml/min for the 3.9 mm and 2.1 mm internal diameter columns respectively. Reequilibration time was calculated using the following formula: [3 x system volume (ml) + 5 x column volume (ml)]/[flow rate (ml/min)] This formula was determined by experimentation with many size columns on different HPLC systems with varying system volumes from 600 µl to several ml. 1 Figure 1: Alkylphenones were separated on 3.9 mm i.d. Symmetry C 18 columns of 150, 50, 20 mm lengths at 1 ml/min. The gradient was 40 to 100% B in 30, 15, and 6 minutes, respectively. AU 3.9 x 20 mm 3.9 x 50 mm 3.9 x 150 mm Minutes

7 Results and Discussion Shorter Columns The use of shorter columns is a very easy way to reduce analysis time. Figure 1 illustrates that the savings in time is inversely proportional to the column length. Using a 2% per minute gradient, the last peak eluted within 25 minutes using the 3.9 x 150 mm column. Although there is some loss in resolution, the alkylphenones are still baseline resolved on the shortest column, 3.9 x 20 mm. This is actually the Waters Sentry guard column, which is packed with the same C 18 material used in the analytical columns. The run time has been reduced from 25 minutes to 5 minutes. Higher Flow Rates A secondary benefit of a shorter column is reduced backpressure. Backpressure decreases in proportion to column length. This allows one to utilise the second strategy for faster chromatography which is higher flow rates. When the flow rates were increased, the gradient run time was shortened in direct proportion to maintain the same number of column volumes across the column, for example, when changing from 1 to 2 ml/min, there is a reduction in the gradient run time from 9 to 4.5 minutes. Figure 2 shows the results of increasing the flow rate on a 3.9 x 50 mm column. The run time can be reduced from less than 9 to less than 3 minutes. Again there is some loss in resolution, but there is still baseline separation of all the peaks. The initial benchmark separation, a 30 minute gradient on a 3.9 x 150 mm at 1 ml/min, has been reduced by an order of magnitude by decreasing the column length and increasing the flow rate. This allows hundreds of samples to be run per day. Change in Resolution How much chromatographic resolution (R) is lost by changing column lengths or flow rates? Figure 3 graphically represents the loss of resolution encountered with the shorter columns and an increase in the flow rate. Resolution was calculated for the last two peaks, 7 and 8. The resolution decreases as a square root function of length and decreases as a linear function of flow rate. Figure 2: Alkylphenones were separated on a 3.9 x 50 mm Symmetry C 18 column at 1, 2 and 3 ml/min. The gradient was % B in 9, 4.5 and 3 minutes, respectively. AU 0 3 ml/min 2 ml/min 1 ml/min Minutes Figure 3: The chromatographic resolution is plotted versus column length for 3.9 mm i.d. or flow rate for 3.9 x 50 mm Symmetry C 18 columns. Resolution x 50 mm Figure 4: Alkylphenones were separated on 2.1 mm i.d. Symmetry C 18 columns of 50 and 30 mm lengths at ml/min. The gradient was % B in 10 and 6 minutes, respectively. AU Column Length mm 2.1 x 30 mm Flow Rate ml/min 2.1 x 50 mm Minutes 7

8 Critical Instrument Performance The overall success of the shorter columns strategy is ultimately determined by the performance of the HPLC system. The critical HPLC system parameters one must consider are the gradient delay volume and the solvent proportioning accuracy at the various flow rates used in this study. The Waters Alliance System has a gradient delay volume of 600 µl. Gradient delay volumes >600 µl result in gradient distortion from a true linear to an S shaped curve. This distortion of the gradient has tremendous impact on the relative retention of the first and last peak. The Waters Alliance System provides the excellent solvent proportioning accuracy required for these fast gradient separations. The standard deviation of all alkylphenone retention times for the fastest separation shown, Figure 5, was minutes (=100). When the column length was shortened to 30 mm, the flow rate could easily be increased 2 or 3 x (Figure 5). Thus the two obvious benefits of this approach are speed and solvent savings. The lower flow rates employed with 2.1 mm columns also make it easier to interface HPLC with a mass detector (LC-MS) and can provide increased sensitivity when compared to 4.6 and 3.9 mm i.d. columns, if the same sample size can be injected. Baseline resolution of all 8 components could now be achieved in under 2 minutes. The Waters Alliance System is a fully integrated HPLC which permits further reduction in the injection-toinjection cycle times and increased throughput. With the Alliance System, the system volume can be automatically purged at a high flow rate, for example 5 ml/min which reduces the total reequilibration time. Conclusion Several ways have been described for obtaining faster gradient separation. They were presented as a series of feasibility studies with model compounds to illustrate how a significant improvement in throughput can be made by considering column dimensions other than the traditional 3.9 x 150 mm or 4.6 x 250 mm. References 1 Li, J., Waters Corporation, unpublished data arrow-bore Column Benefits Speed of the assay is not always the only consideration when developing a method. Method sensitivity and solvent consumption must also be considered. Reducing the consumption of solvent will reduce the cost per analysis. Higher sensitivity is frequently desirable. Lower flow rates are sometimes required for some detection modes like mass detection (LC-MS). The use of narrow-bore columns with a 2 to 3 mm i.d., can meet these needs. Figure 4 repeats the separations of acetone and the seven alkylphenones on 2.1 mm Symmetry C 18 columns (3.5 micron packing), 30 and 50 mm lengths. The smaller particle size provided excellent separation on these short columns. However, a problem was encountered when the flow rate was increased on the 50 mm column. The maximum pressure of the Alliance System was exceeded before the flow rate was increased threefold because of the combinations of the smaller particle size packing and high viscosity eluent (methanol/water) at 30 C. Increasing the temperature to 50 C decreased the viscosity and reduced the backpressure to a usable range (data not shown). Figure 5: Alkylphenones were separated on a 2.1 x 30 mm Symmetry C 18 column at 0.29, 0.58 and 0.87 ml/min. The gradient was % B in 6, 3 and 2 minutes, respectively. AU 0.87 ml/min 0.58 ml/min ml/min Minutes 8

9 Recent Symmetry References Crozier, Alan; et al, Quantitative analysis of the flavonoid content of commercial tomatoes, onions, lettuce and celery, J. Agric. Food Chem. 1997, Vol 45, p 590 Crozier, Alan; et al, Quantitative analysis of flavonoids by reversed-phase high-performance liquid chromatography, J. Chromatogr. 1997, Vol 45, p 315 Freiermuth, Michel; et al, Determination of morphine and codeine in plasma by HPLC following solid-phase extraction, J. Pharm. Biomed. Anal. 1997, Vol 15, p 759 Gaillard, Yvan; et al, Screening and identification of drugs in human hair by high-performance liquid chromatographyphotodiode-array UV detection and gas chromatography-mass spectrometry after solid-phase extraction, J. Chromatogr. 1997, Vol 762, p 251 Hogenboom, A.C.; Rapid analysis of organic microcontaminants in environmental water samples by trace enrichment and liquid chromatography on a single, short column, J. Chromatogr. 1997, Vol 759, p 55 Mendoza, C.B.; et al, Quantitation of an orally available thrombin inhibitor in rat, monkey and human plasma and in human urine by high-performance liquid chromatography and fluorescent post-column derivatization of arginine, J. Chromatogr. 1997, Vol 762, p 299 icholls, Andrew W.; et al, MR and HPLC- MR spectroscopic studies of futile deacetylation in paracetamol metabolites in rat and man, J. Pharm. Biomed. Anal. 1997, Vol 15, p 901 Poirier, Jean-Marie, A rapid and specific liquid chromatographic assay for the determination of itraconazole and hydroxyitraconazole in plasma, Ther. Drug Monit. 1997, Vol 19(2), p 247 Rosseel, M.T.; et al, Measurement of cefuroxime in human bronchoalveolar lavage fluid by highperformance liquid chromatography after solid-phase extraction, J. Chromatogr. 1997, Vol 689, p 438 Shim, Hyun Joo; et al, Determination of a new non-narcotic analgesic, DA 5018, in plasma, urine and bile by high-performance liquid chromatography, J. Chromatogr. 1997, Vol 689, p 422 Sykora, David; et al, Interactions of basic compounds in reversed-phase high-performance liquid chromatography. Influence of sorbent character, mobile phase compostion, and ph on retention of basic compounds, J. Chromatogr. 1997, Vol 758, p 37 Woolf, E.; et al, Determination of an in vivo metabolite of a human immunodeficiency virus protease inhibitor in human plasma by highperformance liquid chromatography wih tandem mass spectrometry, J. Chromatogr. 1997, Vol 762, p 311 Ordering Information Symmetry 3.5 µm Columns Dimension C 18 C mm x 150 mm WAT WAT mm x 30 mm WAT WAT mm x 50 mm WAT WAT mm x100 mm WAT WAT mm x150 mm WAT WAT mm x 50 mm WAT WAT mm x 75 mm WAT WAT mm x 100 mm WAT WAT mm x 150 mm WAT WAT Symmetry 3.5 µm Cartridge Columns* Dimension C 18 C mm x 75 mm WAT WAT mm x 100 mm WAT WAT Symmetry 5 µm Columns Dimension C 18 C mm x 150 mm WAT WAT mm x 150 mm WAT WAT mm x 150 mm WAT WAT mm x 150 mm WAT WAT mm x 250 mm WAT WAT Symmetry 5 µm Cartridge Columns* Dimension C 18 C mm x 50 mm WAT WAT mm x 150 mm WAT WAT mm x 150 mm WAT WAT mm x 250 mm WAT WAT Description Endfittings Integrated Guard Holder** (for Waters steel cartridge columns only) Universal Guard Holder (for any HPLC column) Sentry Symmetry C 18 guard column Sentry Symmetry C 8 guard column * Require reusable endfittings ** Guard Columns require the appropriate Sentry Guard Holder (3.9 mm x 50 mm cartridge columns must use the Universal Guard Holder) For more information on the complete line of Symmetry columns, please check box 3 on the business reply card. Part o. WAT WAT WAT WAT WAT

10 Simple and Fast Methods Development for HPLC and Sample Preparation Yung-Fong Cheng, Zoubair El Fallah, Uwe D. eue, and Dorothy J. Phillips Introduction The development of assays for drugs and their metabolites from plasma or urine sample matrices is time-consuming, since the optimisation of the HPLC method and the sample preparation are difficult and tedious. It is desirable to have a well thought out strategy for the HPLC method development which can rapidly provide good resolution as well as good peak shapes for a wide range of compounds. In addition, it is beneficial to have a universal solid-phase extraction (SPE) sorbent which can extract very polar compounds, acids, neutrals and bases simultaneously, cut down the number of trial chemistries, and simplify method development for the sample preparation. In this paper, a well-thought-out HPLC method development strategy using ph as a primary tool for the separation will be demonstrated. Also, a simple and rugged SPE method using a universal sorbent for the isolation of sample analytes from a biological matrix will be discussed. Results and Discussion Experimental We used a Waters HPLC system with a 616 Solvent Delivery System, a 717plus Autosampler and a 996 Photodiode Array Detector with the Millennium Chromatography Manager. The column for the developmental studies of the separation was a 3.0 x 150 mm Symmetry C 18 column. Sample preparation studies were accomplished using Waters Oasis HLB extraction cartridges. The final chromatographic method was carried out on a 3.9 x 150 mm Symmetry C 18 column. HPLC Method Development Since the majority of pharmaceutical compounds are ionic or ionisable in nature, the variation of mobile phase ph is a powerful methods development tool in reversed-phase chromatography. ph Figure 1: The Selectivity Prism. ph is the primary tool for the separation, and the traditional solvent selectivity triangle is the fine-tuner for further optimisation of the separation. changes induce larger changes in selectivity than solvent changes. Therefore, we employ the variation of ph as our primary tool in methods development, and we use the solvent selectivity to finetune the separation. This experimental space is depicted as the Selectivity Prism (Figure 1), with ph on the vertical axis and the three common organic modifiers in reversed-phase chromatography as the triangular bottom plate of the prism. In order to fully utilise the power of this approach, it is essential to use a reversed-phase packing material that shows little to no peak-tailing independent of the ph value of the mobile phase. Waters Symmetry columns give good peak shape, for a broad range of compounds, independent of the ph of the mobile phase from ph 2 to 8 (1-3). Therefore, we are able to use the powerful tool of ph to establish the basis of the separation first, then use the traditional solvent selectivity approach to further optimise the separation. The foundation of this method development strategy has been reported previously (1,2). Briefly, a solvent is selected from the solvent selectivity triangle, typically methanol or acetonitrile. A gradient is run using this solvent first at a low and than at a high ph value. The chromatograms obtained are compared to each other and the most promising ph is selected for further work. ext, a second gradient is run at the selected ph, which results in information about the retention behavior of the analytes as a function of the solvent composition. Chromatographic theory is then used to select an isocratic solvent composition that results in the correct retention time window. If the separation is not adequate, solvent selectivity is explored to improve the separation, using first methanol and acetonitrile. 10

11 If sufficient selectivity differences are observed, the separation is fine-tuned using isoeluotropic mixtures of both solvents. If this strategy is unsuccessful, THF is used as the third optimisation solvent. This strategy is a good example of the class of strategies that approach the experimental space in steps that are initially very large and subsequently become smaller as a solution to the problem begins to appear. In this article, we present another simple example that demonstrates the power of this strategy using a Symmetry C 18 column. The sample consists of chlordiazepoxide and its metabolites. The nature of these compounds ranges from basic to acidic (structures shown in Figure 2), which demonstrates a good example for using ph as the primary tool in HPLC methods development. We first ran two gradients, at acidic ph 2 and at neutral ph 7, using acetonitrile as the organic modifier and phosphate as the buffer component. Figure 3 shows the effect of ph with acetonitrile gradients (1%/min) at ph 2.0 and ph 7.0, both with 20 mm phosphate buffer. As expected, we observed a significantly different selectivity between acidic ph and neutral ph. At ph 7, all five components are well separated, and nordiazepam elutes last. At ph 2, it coelutes with norchlordiazepoxide. Demoxepam (peak 1 at ph 7) and oxazepam (peak 3 at ph 7) elute at nearly the same time independent of ph. Chlordiazepoxide moves from fourth position at ph 7 to third position at ph 2. Due to the better peak spacing and the fact that all compounds were resolved at ph 7, our next gradient run was performed at ph 7.0 with a gradient slope of 2%/min. This procedure allows us to calculate the isocratic composition that would give us the desired retention time. (1,2) Figure 4A shows the initial isocratic run at 32% acetonitrile. Unfortunately, peaks 1 and 2 are only marginally resolved. We then used the solvent selectivity triangle, the second dimension of the Selectivity Prism, to improve the separation. We used methanol as our first alternative organic solvent and Figure 2: Structure of chlordiazepoxide and its metabolites. The nature of these compounds range from acidic to basic. H 2 O orchlordiazepoxide Chlordiazepoxide (pk a 4.8) Oxazepam (pk a 1.7 and 11.6) O HCH 3 H O OH H Demoxepam (I. S.) ordiazepam O O Figure 3: Effect of ph in acetonitrile gradient runs on the separation of chlordiazepoxide and its metabolites. 1 ph 7.0 ph Retention time (min.) Retention time (min.) 2, unknown H O Column: Symmetry C 18, 3.0 x150 mm Temperature: Ambient Gradient: from 20% MeC/80% 20 mm potassium phosphate to 60% MeC/40% 20 mm buffer Flow rate: 0.59 ml/min Detection: UV at 240 nm 1. Demoxepam 2. orchlordiazepoxide 3. Oxazepam 4. Chlordiazepoxide 5. ordiazepam 11

12 optimised the separation on the bottom plate of the selectivity prism. Based on the theory of isoelutropic elution (1,4), we estimated that 59% methanol should give us about the same retention time as 32% acetonitrile (Figure 4B). By going from acetonitrile as the organic modifier to methanol, peak 2 moves from a near coelution with peak 1 to a coelution with peak 3. Based upon these two figures (Figure 4A and 4B), it is clear that we need a mixture of methanol and acetonitrile to fine tune the separation. With further optimisation of the composition of these two solvents, we obtained optimal separation (at 23% acetonitrile and 28% methanol) as shown in Figure 4C. Within 15 minutes, all five components are well resolved, and they all have excellent peak shapes: the USP plates are all greater than 5,600, and USP tailing factor are all less than 1.22 (Table 1). Simple Solid Phase Extraction (SPE) Method Reversed-phase silica-based sorbents are the most widely used packings for SPE. However, there are three main limitations: For polar analytes, the retention is weak and often results in breakthrough during the loading step. Basic analytes interact strongly with the residual silanols, which in turn cause low recovery. The sorbent must remain wet prior to sample loading. If one accidentally lets the cartridges run dry, the consequence is low and variable recovery. Recently, Waters introduced Oasis HLB extraction cartridges containing a waterwettable sorbent which can overcome all the problems associated with traditional silica-based reversed-phase sorbents (5-7). The Oasis HLB sorbent is a polymeric reversed-phase sorbent. Figure 4: Isocratic separation of chlordiazepoxide and its metabolites at ph 7.0. A: Acetonitrile isocratic run based on three gradient runs. B: Methanol isocratic run based on isoeluotropic series. C: Final isocratic run based on solvent mixture optimisation. Column: Symmetry C 18, 3.0 x 150 mm Temperature: Ambient Mobile phase: See labels Flow rate: 0.59 ml/min Detection: UV at 240 nm A: ph 7.0, 32% MeC B: ph 7.0, 59% MeOH C: ph 7.0, 23% MeC 28% MeOH , unknown unknown Retention time (min.) Retention time (min.) Retention time (min.) 3 5 unknown Table 1: Performance of Waters Symmetry C 18 columns at ph 7.0 for the separation of chlordiazepoxide and its metabolites. USP Plates 1. Demoxepam 2. orchlordiazepoxide 3. Oxazepam 4. Chlordiazepoxide 5. ordiazepam USP Tailing orchlordiazepoxide 5, Oxazepam 6, ordiazepam 6, Chlordiazepoxide 7, Demoxepam 8,

13 This macroporous polymeric packing contains both hydrophilic monomer units (-vinylpyrrolidone) and hydrophobic monomer units (divinylbenzene) (Figure 5). These two unique and distinct properties are carefully balanced. The lipophilic monomer provides the reversed-phase property for analyte retention, while the hydrophilic monomer provides the correct wetting property to keep the surface of the packing solvated even when the cartridges run dry. Furthermore, the Oasis HLB sorbent contains no residual silanols to complicate the interaction with basic analytes. This simplifies the retention mechanism between the analytes and the sorbent. As a result, we can use exactly the same SPE method to extract a wide range of compounds including acids, neutrals, bases, and a wide polarity of analytes (parent drugs and their metabolites) (5-7). For example, here we apply this general SPE method (outlined in Figure 6) to extract chlordiazepoxide and its metabolites from a porcine serum matrix. It is important to point out again that the nature of these compounds ranges from acidic to basic. The general SPE method of Figure 6 is designed to capture a broad range of analytes from serum or plasma samples. Due to the interferences from the serum matrix, further optimisation of the HPLC separation (by varying the ratio of methanol and acetonitrile) was needed to separate the analytes of interest from endogenous interferences present in the matrix blank. The final optimal condition was at 21% acetonitrile and 23% methanol (compared to 23 % acetonitrile and 28% methanol in the preliminary study). Representative chromatograms are shown in Figure 7. The serum blank is shown in Curve A, and the spiked drugs in the serum matrix at a concentration of µg/ml of each analyte are shown in Curve B. The elution sequence is demoxepam (peak 1), norchlordiazepoxide (peak 2),oxazepam (peak 3), chlordiazepoxide (peak 4), and nordiazepam (peak 5). Demoxepam was used as the internal standard. Figure 5: ovel reversed-phase sorbent: the Oasis HLB extraction sorbent. Hydrophilic monomer Regardless of the nature of analytes, when using this streamlined SPE method, we have eliminated time-consuming method development and method refinement studies; samples can be analysed immediately after they arrive in the laboratories. With this general SPE method (outlined in Figure 6), we obtained excellent recoveries with good reproducibility even when we let the cartridges run dry at any stage of the SPE method. The results are summarised in Table 2. For six replicate analyses, the recoveries were all greater than 90%, and the RSDs were all less than 5.5%. Conclusion Symmetry reversed-phase columns give good peak shapes for a broad range of compounds independent of the ph (ph 2 to 8) of the mobile phase. This enables us to use the full ph range accessible to silica-based columns to manipulate the selectivity of a separation. We combined this advantage of Symmetry packings with a rational method development strategy (the Selectivity Prism ) that allows us to develop rugged HPLC methods fast. O water loving Provides the correct wetting property to keep the surface of the packing solvated even when the cartridges run dry Lipophilic monomer fat loving Provides reversed-phase property for analyte retention Figure 6: Schematic of a simple, fast, rugged, and highly reproducible solidphase extraction method using the Oasis HLB 1 cc/30 mg extraction cartridges. With this general SPE method, we are able to extract a wide range of compounds including acids, neutrals, and bases from a porcine serum matrix. We use the same simple method to extract both parent drugs and their extreme polar metabolites from biological matrices. Condition 5 ml methanol Equilibrate 1 ml water Load 1 ml spiked sample Solution Wash 1 ml 5% methanol in water Elute 1 ml methanol Evaporate and Reconstitute 13

14 Oasis HLB sorbent is a universal sorbent. There are no residual silanols to complicate the interaction between the sample analytes, especially basic analytes, and the sorbent itself. With one simple and general SPE method, we are able to extract a wide polarity of compounds with excellent recoveries and reproducibility. Moreover, we can let the Oasis HLB extraction cartridges run dry before loading the sample solution. The time-consuming and tedious manipulation of stopcocks is no longer necessary. This makes SPE more efficient, more rugged, and less tedious. Also, it results in higher sample throughput. In summary, the combination of the Selectivity Prism for HPLC separation using Symmetry columns and the general SPE method using Oasis HLB extraction cartridges can simplify methods development of drugs and their metabolites to an extraordinary degree. References Bouvier, E.S.P.; Martin, D.M.; Iraneta, P.C.; Capparella, M.; Cheng, Y.F.; Phillips, D.J. LC GC 1997, 15(2) p 152 Cheng, Y.F.; Phillips, D. J. J. Liquid Chromatogr. 1997, 20(15), p 2473 Cheng, Y.F.; Phillis, D. J. Chromatographia 1997, 44 (3/4), p 187 El Fallah, M.Z.; In HPLC Columns, Technology, Theory and Practical Use, eue, U.D.; Wiley-Interscience: ew York, Y, 1997, El Fallah, M.Z. Waters Column 1996, Vol. VI(1), p 1 Table 2: Recoveries and reproducibility of chlordiazepoxide and its metabolites using the general method (as outlined in Figure 6). Results of six replicate analyses from porcine serum matrices. orchlordiazepoxide Oxazepam ordiazepam Chlordiazepoxide Figure 7: Chromatograms of (a) serum blank and (b) serum spiked with chlordiazepoxide and its metabolites. AU Concentration Recovery RSD (µg/ml) (%) (% n=6) Column: Symmetry C 18, 5 µm,3.9 x150 mm Mobile Phase: 20mM phosphate: ph 7: acetonitrile: methanol (56:21:23) Detection: UV at 240 nm Flow rate: 1.0 ml/min Injection Volume: 20 µl 1. Demoxepam 2. orchlordiazepoxide 3. Oxazepam 4. Chlordiazepoxide 5. ordiazepam 4 5 eue, U.D.; Phillips, D.J.; Young, M.S.; Walter, T.H.; O Gara, J.E.;Capparella, M.; Alden, B.A. Waters Column 1997, Vol. VI(4), p 8 Schoenmakers, P.J. Optimization of Chromatographic Selectivity, J. Chromatography Library, Vol. 35, Elsevier: Amsterdam, B A Minute 14

15 Oasis HLB Sample Extraction Products For more information on Oasis HLB Sample Extraction products, please check box 5 on the business reply card. Waters Oasis HLB extraction cartridges and 96-well extraction plate contain a unique (patent pending) sorbent, a copolymer designed to have a hydrophilic-lipophilic balance (HLB), that gives high and reproducible recoveries for acidic, basic, and neutral compounds even if the sorbent runs dry. Ordering information Oasis HLB Sample Extraction Products Description Quantity Part umber Oasis HLB extraction cartridge 1 cc/30 mg 100/box WAT Oasis HLB extraction cartridge 3 cc/60 mg 100/box WAT Oasis HLB extraction plate 30 mg/96-well 1/pkg WAT Oasis HLB extraction plate 30 mg/96-well 3/pkg WAT Manifold for Extraction Plate For your free copy of the Oasis HLB Extraction Cartridges Applications otebook, please check box 6 on the business reply card. Extraction Plate Manifold Kit A WAT (includes extraction plate manifold, reservoir tray, manifold top gasket, sealing cap and 350 µl sample collection plate) Extraction Plate Manifold Kit B WAT (includes extraction plate manifold, reservoir tray, manifold top gasket, sealing cap and 1 ml sample collection plate) Extraction Plate Manifold Kit C WAT (includes extraction plate manifold, reservoir tray, manifold top gasket, sealing cap and 2 ml sample collection plate) Extraction plate manifold 1/box WAT Reservoir tray 25/box WAT Sample collection plate 350 µl 50/box WAT Sample collection plate 1 ml 50/box WAT Sample collection plate 2 ml 50/box WAT Sealing cap for 96-well collection plate 50 sheets/pkg WAT Manifold gasket, top 1/pkg WAT Manifold gasket, white 1/pkg WAT Manifold for Extraction Cartridges Waters extraction manifold, 20-position (complete with rack for 13 mm x 75 mm tubes) Waters extraction manifold, 20-position (complete with rack for 13 mm x 100 mm tubes) Waters extraction manifold, 20-position (complete with rack for 16 mm x 75 mm tubes) Waters extraction manifold, 20-position (complete with rack for 16 mm x 100 mm tubes) Vacuum pump (110V, 60 Hz) Vacuum pump (220V, 50 Hz) Vacuum pump (110V, 50 Hz) WAT WAT WAT WAT WAT WAT WAT

16 What s ew ew Symmetry300 C 18 Column For information on Waters Symmetry300 columns, see our web site at Waters continues to expand the Symmetry column product line with the introduction of Symmetry300 C 18 columns extending the standard of reproducibility to proteins and peptide assays. Are you a research chemist developing HPLC assays for well characterised biotechnology products? proteins? peptides? ow available Symmetry300, a wide-pore 300Å reversed-phase HPLC column that meets your need for developing robust and rugged methods that satisfy the ICH method validation requirements. Continuing to set the industry standard for batch-to-batch reproducibility, Waters employs a tryptic map as the key quality control test for the Symmetry300 column including acceptance criteria of retention time and critical pair resolution. ow batch-to-batch or column-to-column variability has been reduced to the point where it is undetectable within the normal test uncertainty of your HPLC method. ew Symmetry Short Columns See page 10 for complete ordering information of these columns. To maximise an assay s sensitivity and speed, a high efficiency, short, narrow-bore or microbore column should be chosen. With these considerations in mind, Waters has developed a new family of Symmetry HPLC columns that meet the method ruggedness requirements for the assays typically performed during a run, and allow you to maximise the speed and sensitivity of LC/MS/MS assays. These new columns are packed with Symmetry 3.5 µm C 18, C 8 or SymmetryShield RP µm packings. The physical characteristics of these new columns were selected after extensive collaboration and consultation with scientists in the pharmaceutical industry who routinely use LC/MS/MS for drug and metabolite analysis from biological fluids. Waters Sep-Pak Solid-Phase Extraction Cartridges For more information, call Waters is celebrating the 20th anniversary of the introduction of Sep-Pak cartridges, the first compact, convenient, silica-based solid-phase extraction device for sample preparation in the market. Waters Sep-Pak cartridges catalysed the establishment of solid-phase extraction as a preferred method in sample preparation. It started in January 1978 with the introduction of the original Sep-Pak cartridges followed by Sep-Pak Plus and then Sep-Pak Light cartridges. Waters later introduced the Sep-Pak Vac syringe barrel cartridge line in a wide choice of cartridge sizes (1 cc 35 cc) and sorbent weights ranging from 50 mg up to 10 grams. All these devices are available in a wide selection of chemistries and are designed for both manual and automated use. 16

17 HPLC Columns published by John Wiley & Sons Theory, Technology and Practice Uwe D. eue, Waters Corporation, Milford, MA For U.S. orders: Phone: call toll free, CALL-WILEY Fax: For European orders: Phone: UK only, Overseas Fax: +44 (0) High performance liquid chromatography and its derivative techniques have become the dominant analytical separation tools in the pharmaceutical, chemical, and food industries, environmental laboratories, and therapeutic drug monitoring. Although the column is the heart of the HPLC instrument and essential to its success, until now no book has focused on the theory and practice of column technology. HPLC Columns provides thorough, state-of-the-art coverage of HPLC column technology for the practicing technician and academician alike. Along with a comprehensive discussion of the chemical and physical processes of the HPLC column, it includes fundamental principles, separation mechanisms, available technologies, column selection criteria, and special techniques. Special features include: Explanation of the underlying principles of HPLC columns Methods for selecting columns Practical advice on using and applying columns, including examples Section by M. Zoubair El Fallah on methods development Special techniques, including preparative chromatography, continuous chromatography, and the simulated moving bed Troubleshooting section Education and Training Services ow Known As Connections SM University For information on Connections University visit our web site at Fax us at or call Waters is dedicated to providing the support to customers that has enabled them to take advantage of our expertise in the areas of HPLC instrumentation, data, sample cleanup and column chemistry. Connections SM University provides certification in understanding the chromatographic process. Consider attending the generic courses offered on topics of column chemistry, method validations and developing HPLC separations. The course Chemistry of Resolution will assist people utilising chromatography in learning about the effects of base silica, porosity, column dimensions and more as these affect separations, sensitivity and run time. Courses on method development and method validation strategy will guide you toward the development of rugged, robust separations that will aid you in meeting regulatory requirements. 17