Fast and Easy Solid-Phase Extraction with a New Reversed-Phase Sorbent

Volume VI Issue 4, 997 Fast and Easy Solid-Phase Extraction with a New Reversed-Phase Sorbent Dorothy J. Phillips, Laura L. Bean, Edouard S.P. Bouvier, Mark Capparella, Yung-Fong Cheng, Pamela C. Iraneta and Uwe D. Neue biological fluids, is now the bottleneck and needs to be automated. But liquid-liquid extraction and protein precipitation methods do not lend themselves to automation. The method of choice is solid-phase extraction (SPE). Using robotics and miniaturisation has enabled GlaxoWellcome to process 50,000 samples a day (). Many other pharmaceutical companies are struggling to increase sample throughput with vacuum manifolds and robotic sample processors. Table of Contents Waters asis HLB extraction cartridge the most important advance in solid-phase extraction in 0 years. The Challenge ver the past five years, pharmaceutical companies have made a fundamental change in the way they conduct their drug discovery programs. Their preferred discipline now is combinatorial chemistry. Combinatorial chemistry integrates such tools as novel synthesis, reaction equipment and advanced information systems to accelerate the generation and optimisation of pharmaceutical candidates (). The ultimate vision for a combinatorial or high throughput chemistry laboratory is the synthesis of millions of individual, pure compounds each day (). The implementation of combinatorial chemistry in the discovery programs has resulted in an increase in the number of drug candidates in the preclinical phase. Drug metabolism groups now have many more candidates to screen and need to work even faster to shorten the drug discovery cycle. Hence, there is a critical need for more efficient methods to extract and analyze samples from biological fluids. The analytical technique that is stepping up to the challenge is LC/MS/MS which can analyse samples at a rate of one sample every 5 minutes. Sample preparation, required for the analysis of most drugs in Solid-Phase Extraction...-7 Improving Method Development...8- Evaluation of Imitation µbondapak Columns...-5 How to Reach Us...5 Papers Presented at HPLC 97...6-7 What s New...8 Application Notes...9- Androgens: Testosterone Esters Morphine and Its Metabolites Editor: Uwe D. Neue, Ph. D. ISSN # 084-0540 997 Waters Corporation

Traditional Problems Development and implementation of solid-phase extraction methods can be tedious. It is a challenge to devise methods that deliver high recoveries reproducibly. For at least three reasons, traditional SPE products, whether used with manual vacuum manifolds or robotic systems, often do not achieve this goal. First, the silica-based reversed-phase sorbents in these traditional SPE products weakly retain the more polar drugs and metabolites. Such weak retention causes sample breakthrough yielding unpredictably low recoveries. Second, optimising a method for reproducible recoveries can be timeconsuming because traditional sorbents contain a heterogeneous population of surface silanol groups which have a strong and variable retentive effect on basic analytes. Finally, perhaps the major contributor to irreproducibly low recoveries is the inability of the traditional silica- and polymer-based reversed-phase sorbents to be wetted with water. If these sorbents are allowed to run dry at any time before the sample loading step in an SPE protocol, only a fraction of the sorbent s available surface will be wetted by an aqueous sample. Adsorption capacity for drugs and metabolites will thereby be reduced, resulting in low, variable recoveries (,4). A Unique Solution Waters new asis HLB extraction cartridges overcome these limitations of traditional silica- and polymer-based reversed-phase SPE products (,4). asis HLB extraction cartridges contain a novel polymeric sorbent (patent pending) that makes it fast and easy to develop reproducible extraction methods yielding high recoveries. The simple reversed-phase extraction mechanism of this unique new sorbent offers stronger, more predictable retention of polar drugs and metabolites, including basic compounds. But most important, asis HLB extraction cartridges eliminate the major cause of variability in solid-phase extraction. Unlike traditional hydrophobic reversedphase sorbents, asis HLB sorbent is fully wettable with water. This means there is no longer a need to monitor vacuum levels, program robotic adjustments, or twist stopcocks to keep sorbent beds from drying out during the critical cartridge conditioning, equilibration, and sample loading steps. asis HLB sorbent is a macroporous copolymer (poly(divinylbenzene-co-nvinylpyrrolidone) composed of both lipophilic and hydrophilic monomer units. This hydrophilic-lipophilic balance gives asis HLB sorbent its unique reversed-phase extraction performance advantages (and its name): High and reproducible recoveries for acidic, basic and neutral drugs with a wide range of polarities. Simple and rapid methods development for drugs and metabolites. Easily automated methods for high sample throughput. Streamlined manual or automated analyses since the sorbent can run dry during the process without affecting the reproducibility of results. Bouvier et al. discussed the theory and advantages of the asis HLB sorbent s water wettability in earlier papers (, 4). In this paper, we report the extraction of drugs from serum using asis HLB extraction cartridges. A simple SPE method gave reproducibly high recoveries for acidic and basic drugs of varying polarity. Experimental Reagents and Materials Salicylic acid, benzoic acid, propranolol, dipropyl phthalate and butylparaben were purchased from Aldrich Chemical Company (Milwaukee, WI). Procainamide, ranitidine, acetaminophen, sulfanilamide, propranolol, doxepin hydrochloride, betamethasone valerate, minocycline, tetracycline and demeclocycline were obtained from Sigma Chemical Company (St. Louis, M). Acetonitrile, methanol, potassium phosphate, sodium phosphate, sodium chloride, glacial acetic acid and phosphoric acid were obtained from J. T. Baker (Phillipsburg, NJ). Porcine serum was obtained from Equitech-Bio (Ingram, TX). asis HLB extraction cartridges, cc/0 mg were obtained from Waters Corporation (Milford, MA). Bond Elut C 8, cc/00 mg solid-phase extraction (SPE) cartridges were purchased from Varian Sample Preparation Products (Harbor City, CA). Solid-Phase Extraction of Drugs in Serum Sample pretreatment for the tetracyclines, salicylic acid and benzoic acid involved adding 0 µl of phosphoric acid to ml of spiked porcine serum before sample loading. Figure outlines the general solid-phase extraction procedure developed on the asis HLB extraction cartridges (). The cartridges were placed on a 0 position Waters Extraction Manifold attached to a Waters vacuum pump. The cartridges were conditioned with ml methanol and equilibrated with ml water; the specific flow rate was not important for these steps. Then a ml serum sample containing the drug(s) was added at a flow rate of < ml/min (vacuum level ~ -4 Hg). The cartridges were then washed with 5% methanol in water to remove polar interferences. The analyte was eluted at a flow rate of < ml/min with methanol. Figure : General SPE method developed for analysis of drugs in biofluids using asis HLB extraction cartridges. Condition ml methanol Equilibrate ml water Load ml spiked serum Wash ml 5% methanol in water Elute ml methanol Evaporate and Reconstitute 40 C /00 µl mobile phase 997 Waters Corporation

Finally, the methanol eluate was evaporated to dryness at 40 C under a nitrogen stream, and the sample was reconstituted in mobile phase at the desired concentration. Flow through the asis HLB extraction cartridges did not have to be monitored because the cartridges can run dry without loss of recovery or reproducibility. In each study, a pair of porcine serum samples were prepared, each containing a different drug level, and then each sample was extracted in replicates of six, using an appropriate internal standard as indicated. For the tetracyclines study, the first serum sample contained tetracycline and minocycline each at.5 µg/ml; in the second sample, the concentrations of each were 0.5 µg/ml. Demeclocycline was added to both serum samples as an internal standard at 5.0 µg/ml. Similarly, for the acids experiments, salicyclic and benzoic acids were added to a pair of porcine serum samples at respective drug levels of µg/ml and 5 µg/ml. In this case, the internal standard, nalidixic acid at an effective level of 5 µg/ml, was added to the analyte-containing fraction from each extracted sample after its evaporation and reconstitution in mobile phase. The same protocol above was repeated using the Bond Elut C 8 cartridges, with only one critical difference: with these silica-based C 8 cartridges, the entire SPE protocol was monitored closely to prevent cartridge drying, especially during the conditioning, equilibration, and sample loading steps. Batch Reproducibility Study Phosphate-buffered saline was prepared containing the following amounts of anhydrous salts per liter of water: a) 00 mg potassium chloride, b) 8000 mg sodium chloride, c) 00 mg potassium monobasic phosphate and d) 50 mg sodium dibasic phosphate. The solution was adjusted to ph 7.0 with 0% phosphoric acid. Phosphate-buffered saline was spiked with two drug mixtures. ne drug mixture contained procainamide, ranitidine, and acetaminophen; the other mix had propranolol, doxepin, dipropyl phthalate and betamethasone valerate. Solid-phase extraction was done with six replicates for each drug mixture on each of three batches of asis HLB sorbent. The SPE procedure in Figure was used. The internal standard (0 µl of either sulfanilamide or butyl paraben at mg/ml, respectively) was added after reconstitution of the appropriate eluate fraction. HPLC Analysis of Drugs The analyses used various Waters HPLC instruments equipped with Waters 77plus Autosamplers and the Millennium 00 Chromatography Manager (Waters Corporation, Milford, MA), either a Waters 65 LC System or a Waters 66 Multi Solvent Delivery System, and either a Waters 486 Tunable Absorbance Detector or a Waters 996 Photodiode Array Detector. A Spark Holland Mistral Column ven (Euramark, Mount Prospect, IL) was used to control column temperature. For analysis of tetracycline and minocycline, a Waters column (.0 x 50 mm) coupled with a Sentry guard column was used at room temperature. The mobile phase consisted of 0.% TFA in water/methanol/acetonitrile 9//7 (v/v/v). Flow rate was 0.9 ml/min; detection was at 50 nm. The injection amount was 0 µl of reconstituted porcine serum extract. Salicylic and benzoic acid were analysed on a Waters column (.0 x 50 mm) coupled with a Sentry guard column; the column temperature was 40 C. The mobile phase was.5% glacial acetic acid/acetonitrile 77: (v/v). Flow rate was 0.6 ml/min; detection was at 6 nm. The injection volume was 0 µl. Procainamide, ranitidine and acetaminophen were analysed on the Waters column (.9 x 50 mm) coupled with a Sentry guard column. The temperature was controlled to 0 C. The mobile phase was 0 mm potassium phosphate, ph /methanol 97: (v/v). Flow rate was.0 ml/min; detection was at 0 nm. The injection volume was 50 µl. Doxepin, propranolol and betamethasone valerate were analysed on a Waters Symmetry C 8 column (.9 x 50 mm) with a Sentry Symmetry C 8 guard column. The mobile phase was 0 mm potassium phosphate buffer, ph 7/methanol 5:65 (v/v). Flow rate was.0 ml/min; detection was at 54 nm. The injection volume was 0 µl. Results The simple, rugged, and highly reproducible solid-phase extraction (SPE) method that was developed for the asis HLB extraction cartridges is outlined in Figure (). Due to the simplicity of the interaction of analytes with the asis HLB sorbent, it was possible to develop a general SPE method for extraction of many kinds of drugs and their metabolites from serum and plasma. This SPE method is applicable to a wide range of analytes; it can be modified or fine tuned to remove specific interferences or to extract compounds at the extremes of polarity (very hydrophilic or hydrophobic drugs). Tetracyclines ne of the causes for low and variable recovery on the traditional silica-based reversed-phase cartridges is the strong retention of basic compounds by ionic interaction with deprotonated silanols. In addition, metals present in the silica packings can also retain drugs, such as tetracyclines, which are metal chelators. Higher and more consistent recoveries of such compounds would be expected from the asis HLB extraction cartridges which do not contain either silanols or metals. Indeed, when the amphoteric tetracyclines were extracted from serum using asis HLB extraction cartridges and the simple general SPE method, recovery and reproducibility were found to be significantly better than the results obtained when C 8 -silica SPE cartridges were used (see Table ) (5). The structures of the tetracyclines used in this study are shown in Figure. Under typical SPE conditions, tetracyclines are amphoteric and can chelate with metals. The results in Table show that optimum extraction of the tetracyclines is achieved if the spiked serum is pretreated with acid before loading on the cartridges. 997 Waters Corporation

The tetracyclines are known to bind to serum proteins; the literature reports up to 75% retention of tetracyclines by serum proteins (6, 7). The three acids, % phosphoric acid, % TFA and 6 mm citric acid, were equally effective and resulted in essentially 00% recovery of minocycline and tetracycline. All three acids were able to disrupt the drug-protein interaction; phosphoric acid was selected for reasons of safety and convenience. The chromatograms in Figure of the blank serum (A) and the extracted drugspiked sample (B) show that no endogenous peaks interfere with the HPLC analysis of the drugs or the internal standard. Choosing an absorption maximum wavelength of 70 or 50 nm allows detection of the tetracyclines where few endogenous compounds absorb. The excellent peak shapes on the column also facilitated quantitation with high accuracy and precision. Extractions using the asis HLB extraction cartridges with our general method gave less than.5% RSDs and greater than 95% recoveries for two levels of minocycline and tetracycline; the data for both the.5 and 0.5 µg/ml levels are in Table. With the method outlined in Figure, results obtained using asis HLB extraction cartridges were compared to those using the silica-based Bond Elut C 8 cartridges. Table lists 40.7% and 67.4% recoveries from the Bond Elut C 8 cartridges versus 94.8% and 04% recoveries from the asis HLB extraction cartridges for minocycline and tetracycline, respectively. The reason(s) for the inferior recoveries obtained by SPE with the C 8 cartridges are not clearly understood. Interactions of tetracyclines with the surface of silica-based reversed-phase sorbents are typically manifested in chromatograms by tailing and distorted peaks. In the literature, this behavior has been attributed to silanol interaction and/or complexing with metal contaminants. These complications clearly do not occur with the unique polymer sorbent in asis HLB extraction cartridges. Table : Comparison of SPE results for tetracyclines. asis HLB Extraction Cartridge C 8 Cartridge Concentration Recovery RSD Recovery RSD (µg/ml) (%) (%) (%) (%) Minocycline.5 94.8.4 40.7 0.8 0.5 0 0.54 Tetracycline.5 04 0.55 76.4 0.44 0.5 97.8.4 Table : Recovery of tetracyclines as a function of the type of acid used for sample pretreatment. The SPE method using asis HLB extraction cartridges is described in the text. % % 6 mm Phosphoric Acid Trifluoracetic Acid Citric Acid Minocylcline 98.7% 0% 05% Tetracycline 97.% 06% 0% Figure : Structures of drugs and internal standards. NMe NMe H H H Minocycline H CI CH Tetracycline H H NMe NMe Demeclocycline (I.S.) CNH CNH CNH H C N C Benzoic acid C Salicylic acid CH CH N Nalidixic acid (I.S.) C 997 Waters Corporation 4

Polar Acidic Drugs The asis HLB extraction cartridges retain drugs and their metabolites by the same reversed-phase mechanism as do the traditional silica-based reversedphase cartridges. Acidic and neutral drugs do not interact secondarily with the silanols on the silica-based sorbents. Therefore, these compounds should interact with the Bond Elut C 8 and the asis HLB sorbents in a similar manner. Both cartridge types should show the same high recoveries. The only proviso is the polarity of the drug since the more polar drugs may be poorly retained on the C 8 cartridges. Salicylic and benzoic acids are polar compounds with pk a s close to.0; the structures are shown in Figure. They were extracted by asis HLB extraction cartridges with the same SPE method employed for the amphoteric tetracyclines. Table illustrates the importance of extracting the polar acids in protonated form. When salicylic acid was extracted from spiked saline at ph 7.0 only 6.5% recovery was obtained. In contrast, extraction of these acidic compounds in the protonated form at ph < gave 00% recovery. A protonated acid is less polar than its corresponding ionic form and is therefore better retained on a reversed-phase SPE sorbent. Excellent peak shapes were obtained for both salicylic and benzoic acid on the column which enabled accurate quantitation of these compounds in serum or saline after solidphase extraction. The chromatography is shown in Figure 4 for both the extracted acids and the serum blank. Table 4 shows the recoveries obtained when salicylic acid and benzoic acid at both 5 µg and µg per ml levels were extracted from serum using asis HLB extraction cartridges. Recoveries greater than 85% percent and RSDs less than 5% were obtained. Figure : Chromatograms of (A) serum and (B) serum spiked with minocycline and tetracycline using demeclocycline as internal standard. Chromatographic conditions are described in the text. 0.0 AU 0 0 5 Table : Effect of sample ph during loading on recovery of salicyclic acid. Compounds Concentration Load at ph 7 Load at ph < µg/ml Recovery(%) Recovery (%) Salicylic Acid 0 6.5 0 pk a.97,.4 Figure 4: Chromatograms of (A) serum and (B) serum spiked with salicylic acid and benzoic acid using nalidixic acid as internal standard. Chromatographic conditions are described in the text. 0.05 AU 0 0 5 0 5 : Minocycline : Tetracycline : Demeclocycline (I.S.) 0 5 0 5 0 Column:, 5 µm, mm x 50 mm Mobile phase: Acetonitrile: % glacial acetic acid (75:5) Flow rate: 0.6 ml/min Detection: UV at 6 nm Injection Volume:0 µl Peaks: : Benzoic acid : Nalidixic acid (I.S.) : Salicylic acid B A B A 5 997 Waters Corporation

When the acids were extracted from acidified serum, using Bond Elut C 8 cartridges and the procedure detailed in Figure, being careful not to let the cartridges run dry, the results were similar to those obtained with the asis HLB extraction cartridges. Greater than 90% recoveries were obtained for salicylic acid at µg/ml on both types of cartridges. Because acidic compounds interact with the SPE sorbents used here principally by a reversed-phase (hydrophobic interaction) mechanism, no differences were expected or observed between the results obtained using traditional bonded-silica C 8 cartridges and those using asis HLB extraction cartridges. Many methods that have been developed on silica-based reversed-phase cartridges can be transferred directly to asis HLB extraction cartridges. The key advantage in doing this is that flow through asis HLB extraction cartridges does not have to be monitored. Cartridge drying as described above will not lead to low and/or variable recoveries. Reproducibility A high level of reproducibility is built into the synthetic design and the manufacturing process for the asis HLB sorbent. Each sorbent batch is quality controlled by eleven different tests. Physical property specifications include pore size, particle size, surface area, total pore volume and fines content (% of particle volume with particles less than 0 micron in size). Recoveries and relative standard deviation (n=6) are reported for the solid-phase extraction of three polar drugs procainamide, ranitidine and acetaminophen. A sorbent selectivity test ensures that each batch has identical retention characteristics. This selectivity test involves packing the sorbent in an HPLC column to measure relative retentions and a capacity factor for a mixture of acidic (salicylic acid), basic (ranitidine) and neutral (caffeine, p-toluamide) compounds. Specifications and sorbent batch test results are reported on a Certificate of Analysis which is included in each box of product. As a result of this stringent quality control, excellent batch-to-batch reproducibility has been observed for the asis HLB sorbent. Figure 5 shows the high recovery obtained for seven compounds, ranging from the very polar neutral acetaminophen to the more lipophilic dipropyl phthalate and betamethasone valerate and including basic drugs such as procainamide, propranolol, doxepin and ranitidine. Figure 5: Batch-to-Batch Reproducibility of asis HLB Sorbent in cc/0 mg Cartridges % Recovery 00 80 60 40 0 0 Batch A Batch B Batch C 0.96%.%.0%.66%.40%.06%.67% Procainamide Ranitidine Acetaminophen Regardless of the nature of the drug, greater than 90% recoveries were observed. More important is the fact that the recoveries for all drugs were highly consistent from batch-to-batch. RSDs.67% were obtained across three different batches of asis HLB sorbent. These data along with the low % RSDs for replicates of six cartridges per analysis (Tables and 4) illustrate the high reproducibility from batch-to-batch and cartridge-to-cartridge, respectively, for the asis HLB extraction cartridges. Table 4: Recoveries of salicylic and benzoic acids extracted from serum using asis HLB extraction cartridges. The SPE method is described in the text. Compound Concentration (%) Recovery (%)RSD (µg/ml)) (n=6) Salicylic acid 5.0 9..0 9 5.0 Benzoic acid 5.0 87.0.0 85.8 Propranolol DP-phthalate Doxepin Betamethasone Valerate 997 Waters Corporation 6

Conclusions asis HLB extraction cartridges are a universal replacement for, and provide a solution to the problems associated with, traditional silica-based reversed-phase SPE cartridges. The absence of silanol interaction and the excellent retention of polar drugs on the asis HLB extraction cartridges make it possible to develop ideal sample preparation methods without difficulty. Compounds as different as tetracyclines, basic drugs, and polar acids were all extracted with high recoveries using the same simple, rugged and highly reproducible method. High and consistent recoveries were obtained across cartridges and batches of asis HLB sorbent regardless of an analyte s polarity or chemical functionality. These new reversed-phase SPE cartridges make sample preparation fast and easy for the analytical chemists who are quantitating drugs and metabolites in biological fluids. asis HLB extraction cartridges should replace C 8 cartridges in existing methods and should be the cartridge of choice for developing all new SPE protocols. References. A. A. MacDonald, D. G. Nickell and S. H. DeWitt, Pharmaceutical News, (6) 996 pp 9.. GlaxoWellcome sponsors, Nature, 84 (6604 supplement) 996 pp 6.. E.S.P. Bouvier, D.M. Martin, P.C. Iraneta, M. Capparella, D. Phillips, Y-F. Cheng and L. Bean, Waters Column, VI() 996 pp 5. 4. E.S.P. Bouvier, D.M. Martin, P.C. Iraneta, M. Capparella, Y-F. Cheng and D.J. Phillips, LC:GC, 5() 997 pp 5 58. 5. Y-F. Cheng, D.J. Phillips, and U.D. Neue, Chromatographia, 44(/4) 997 pp 87-90. rdering Information Description Qty Part No. asis HLB Extraction Cartridge cc/0 mg 00/box WAT0945 asis HLB Extraction Cartridge cc/60 mg 00/box WAT0946 To achieve the maximun advantage of the asis HLB extraction cartridge, we recommend the new Waters Extraction manifold (see page 8). NEW asis HLB Extraction Plate The Waters asis HLB Extraction Plate (96-well) is available with 0 mg of sorbent in each well. Each well contains 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 in the wells runs dry. Description Part No. asis HLB Extraction Plate (0 mg/96 well) /pkg WAT05895 asis HLB Extraction Plate (0 mg/96 well) /pkg WAT05899 Extraction Plate Manifold WAT05894 6. H. Kalant and W. H. E. Roschlau, Principles of Medical Pharmacology, B. C. Decker, 989, p. 7. R. C. Heel and G. S. Avery, Drug Data Information in Drug Treatment, Principles and Practice of Clinical Pharmacology and Therapeutics, Adis, 980, pp. For more information on asis HLB Extraction Cartridges and Extraction Plate, please check box 4 on the business reply card. For your free copy of the asis HLB Extraction Cartridges Applications Notebook, please check box on the business reply card. 7 997 Waters Corporation

Improving Method Development Using a New Reversed-Phase High-Performance Liquid Chromatography (HPLC) Column with Unique Selectivity Uwe D. Neue, Dorothy J. Phillips, Michael S. Young, Thomas H. Walter, John E. Gara, Mark Capparella and Bonnie A. Alden Introduction The process of developing HPLC methods is a critical step in the work of an analytical chemist. Frequently, the investigator encounters a situation where the selectivity obtainable on a particular column fails to meet the separation requirements, even after systematic optimisation of the mobile phase (,). When this happens the chromatographer often resorts to use of unusual mobile phase compositions or additional mobile phase additives. In this paper, we demonstrate an alternative approach: when a separation does not respond to simple variations in mobile phase composition, it is often advantageous to change to a column that has significantly different selectivity characteristics. The Packing Material Waters has recently developed a unique patented reversed-phase packing material ( ) possessing selectivity characteristics significantly different from traditional C 8 and C 8 materials (). The modified selectivity arises from the insertion of a carbamate group into the alkylsilane bonded phase (Figure ). The incorporation of polar groups into alkylsilane bonded phases has been shown to significantly modify their selectivity characteristics, due to perturbation of both analyte/stationary phase and mobile phase/stationary phase interactions (-7). These polar functional groups compete with polar analytes for the interaction with the surface silanols. Therefore, one particularly notable feature of all reversed-phases containing embedded polar group is the reduction in both retention and peak tailing of amines. Figure : Structure of the bonded phase H C CH This reduced retention can be attributed to a shielding of the silanols by the embedded polar group. Discussion In the process of methods development, the investigator often encounters a situation where the selectivity obtainable on a particular packing does not entirely match the requirements of the application. In such a situation, the chromatographer usually struggles for extended periods of time with unusual mobile phase compositions or additional mobile phase additives. An alternative approach that has proven very valuable in our laboratory is the use of a column chemistry that provides similar retention characteristics but different selectivity compared to a classical reversed-phase column. The column provides this alternative. The addition of the carbamate group in the bonded phase significantly changes the interaction between polar functional groups in the analyte molecule and the silanol groups present on all bondedphase packings. As a consequence, switching from a standard C 8 or C 8 column to a column can drastically affect the peak spacing and the elution order of analytes without the need to change mobile phase composition. This allows for a rapid comparison between a standard C 8 column and the column. Si H N C (CH ) 7CH The orientation of the carbamate group should be emphasised. It is oriented such that after a hydrolytic loss of the ligand at the carbamate function, an interactively neutral alcohol function is formed. Although careful hydrolysis studies have not revealed any stability issues, we purposely designed the phase to avoid any inadvertent secondary interactions. This is not the case for other packing materials available on the market, which also rely on polar functional groups to prevent the interaction between analytes and surface silanols (7). A selectivity study of was made using simple substituted benzenes as samples. It was found that, generally, the methylene group selectivity was lower on the column than on a standard Symmetry C 8 column. At the same time, the selectivity for polar functional groups was significantly different for both phases. There was a reduced retention for very polar functional groups on the column, especially for amino-, alcoholand amide- functions, and an increased retention for phenols and acids at acidic ph. This indicates that the influence of acidic silanols on the retention of analytes is decreased using the column. An example of the selectivity differences is shown in Figure. 997 Waters Corporation 8

n the column, we find a significant decrease in retention for benzyl alcohol and benzylamine, and a slight increase in retention for phenol and benzoic acid. It is believed that the reason for these selectivity differences is due to the fact that water strongly binds to the polar carbamate group. This tightly bound layer of water prevents interactions between the analytes and the underlying tether function as well as with the surface silanols of the underlying silica. As a consequence, surface silanols do not play as much of a role in the retention of analytes on the column as they do on a standard C 8 column. Also, good peak shapes are obtained for polar analytes, in addition to the differences in selectivity discussed above. Thus, the name of the column is quite appropriate: the surface silanols are shielded from interacting with the polar functional groups of analytes. Such differences in interaction can result in significant shifts in the selectivity of a separation. As mentioned above, the column often yields a separation when mobile phase modification on a C 8 packing has failed to do so. An example is shown in Figure. n the left, the separation of furazolidone impurities is shown on a Symmetry C 8 column. It appears that there is only a single impurity present. However, as shown in the chromatogram on the right, the column cleanly Figure : Selectivity differences between and Symmetry C 8 packings Columns:.9 mm x 50 mm SymmetryShield RP 8, 5 µm and Symmetry C 8 5 µm Mobile phase: 80% 50 mm KH P 4, ph.0, 0% acetonitrile Flow rate: ml/min Detection: Absorbance at 54 nm Symmetry Shield RP 8 V 0 4 V 0 0 5 0 5 separates the impurity into two well resolved peaks. In this case, the selectivity difference between a column and a Symmetry C 8 column is quite striking. Another example of selectivity differences is shown in Figure 4 4 Symmetry C 8 Peaks:. Benzyl alcohol. Phenol. Benzoic acid 4. Benzylamine for the Alberta peptide standards. A significant reduction in retention is observed for these peptides on the column, as compared to the standard Symmetry C 8 column. Figure : Selectivity comparison for furazolidone impurities Column: a. Symmetry C 8 5 µm b., 5 µm.9 mm x 50 mm Mobile Phase: Gradient at %/min acetonitrile starting at 0% acetonitrile/ 90% 0.% acetic acid, ph. Flow Rate: 0.7 ml/min Detection: Absorbance at 70 nm Injection: 0 µl Sample: Furazolidone and its impurities 0.0 0.008 0.006 0.004 0.00 0 0.0 Symmetry C 8 0.008 0.006 0.004 0.00 0 0 5 0 5 0 5 0 0 5 0 5 0 5 0 9 997 Waters Corporation

This reduction in retention is due to a decrease in the interactions of these basic peptides with the column compared to the Symmetry C 8 column. This reduced interaction is completely in line with the expectations outlined above. It appears that the column provides additional separation capabilities for peptide samples where the options for mobile phase manipulation are more limited. A further example in Figure 5 shows the separation of phenoxyacid herbicides and phenols on a column. The separation of all 6 EPA 555 analytes is accomplished without difficulties using this column. The same separation is not possible using standard reversed-phase columns. nce again, the advantage of the different selectivity achievable with the column is well demonstrated for this application. For separations of compounds with differences in the pk a, significant variations in elution order have been observed between a standard C 8 column and the column. An example is the separation of the weak acid, acetaminophen; the strong base, codeine and caffeine at ph (Figure 6). n the Symmetry C 8 column, codeine elutes after acetaminophen. Using the identical mobile phase conditions on a column, significantly different retention times were observed. Most importantly, the elution order of acetaminophen and codeine changed. The basic compound codeine exhibits less retention than acetaminophen on the column, while it elutes after acetaminophen on the Symmetry C 8 column. This is completely in line with the expectations outlined above. In addition to the selectivity differences demonstrated, columns also perform better than standard reversed-phase columns in highly aqueous mobile phases. Figure 4: Difference in selectivity for polar basic peptides AUFS AUFS.0.05 0.0.05 0 5 Symmetry C 8 0 5 0 4 5 5 Gradient: Figure 5: Separation of phenoxyacid herbicides and phenols on a column Column:, 5µm.9 mm x 50 mm Mobile phase: A: Phosphate Buffer ph. B: Acetonitrile Gradient: 90% A to 70% A in 8 min, hold for 7 min, then linear to 40% A at 0 min, then to 0% A at 5 min Flow rate:.0 ml/min Detection: Absorbance at 0 nm (0.0 AUFS) Sample: 500 ml of tap water spiked with 5 µg/l each component, trace enriched by solid-phase extraction (SPE) Injection: 40 µl of SPE eluent diluted : with buffer 4 5 Columns: 5µm and Symmetry C 8, 5µm.9 mm x 50 mm Mobile phase:a. 0.% TFA in water Flow rate: Detection: Injection: 5 B. 0.% TFA in acetonitrile From 0% to 40% B in 0 min, step to 60% B for 5 min 0.8 ml/min Absorbance at 4 nm 9 µl Alberta Peptide Mixture Sample: Ac-Arg-Gly-X-X-Gly-Gly-Leu-Gly-LysAmide- X-X-: : Ala-Gly with free alpha amino group : Gly-Gly 4: Val-Gly : Ala-Gly 5: Val-Val 0 0 40 997 Waters Corporation 0

This improvement arises because the embedded polar functional group in the bonded phase ensures that the packing remains well wetted even in mobile phases containing 00% water. In contrast, in highly aqueous (> 90% water) mobile phases, high coverage C 8 and C 8 columns often show erratic retention (8). This has been attributed to poor wettability of the bonded alkylsilane groups, preventing the partitioning of analyte into the stationary phase (9). Since the packing does not exhibit this problem, it can be used with advantage in the separation of very polar analytes, which require mobile phases with a high water content. These advantageous wetting properties can be readily demonstrated using very polar analytes, such as the antibiotic amoxicillin. The mobile phase consisted of 99.9% water and 0.% glacial acetic acid. Good retention was achieved when the separation of this analyte was run on a Symmetry C 8 column. However, when the flow was stopped for a short time (5 min), then resumed, the retention of this compound was lost completely. It was only possible to regain the original retention of the compound by reconditioning the stationary phase first with methanol, then with the aqueous mobile phase. When the same experiment is performed using a column, no loss of retention is observed. Fundamentally, this shows that the surface remains wetted even when a mobile phase containing 00% water is used. This wetting phenomenon is due to the polar functionality incorporated into the stationary phase. As mentioned already in the discussion of the retention mechanism, the polar carbamate group of the column binds water very strongly, thereby preventing the hydrophobic collapse observed with standard reversed-phase columns in mobile phases with a very high water content. Consequently, the column can be used advantageously for the analysis of very polar compounds that require mobile phase compositions approaching 00% water. Figure 6: Gradient separation of acetaminophen, codeine and caffeine Columns: a. 5 µm.9 mm x 50 mm b. Symmetry C 8 5 µm,.9 mm x 50 mm Mobile phases: A. 50 mm potassium phosphate, ph B. Acetonitrile Gradient: 5% to 50% acetonitrile in 5 min Flow rate: ml/min Detection: Absorbance at 54 nm Injection volume: 5 µl 0 4 Symmetry C 8 0 4 Conclusion The column represents a powerful new tool for reversed-phase separations. The selectivity of this column for compounds with polar functional groups can be significantly different from the selectivity of standard reversed-phase columns. This property can be used advantageously in methods development. This is especially true in circumstances where a good separation with standard reversedphase columns appears to be difficult to achieve. The difference in the selectivity characteristics observed between the column and standard reversed-phase column is primarily due to the shielding of the surface silanols by the carbamate group incorporated in column. Another advantage of this column is its good wettability in highly aqueous mobile phases. The column significantly enhances the tools available to the methods development chemist and opens a new dimension in reversedphase selectivity. 6 8 6 8 References Peaks:. Codeine. Acetaminophen. Caffeine Peaks:. Acetaminophen. Codeine. Caffeine. M.Z. El Fallah, Waters Column, Vol. 6 () 995,. M.Z. El Fallah, in U.D. Neue HPLC Columns Technology, Theory and Practical Use, Wiley-Interscience, New York, 997. J.E. Gara, B.A. Alden, T.H. Walter, J. S. Petersen, C.L. Niederlander and U.D. Neue, Anal. Chem. 67, 995, 809 4. B. Buszewski, J. Schmid, K. Albert and E. Bayer, J. Chromatogr. 55, 99, 45 5. B. Buszewski, M. Jaroniec and R.K. Gilpin, J. Chromatogr. A67, 994, 6. B. Buszewski, M. Jaroniec and R.K. Gilpin, J. Chromatogr. A668, 994, 9 7. T.L. Ascah, K.M.R. Kallury, C.A. Szafranski, S.C. Corman and F. Liu, J. Liq. Chrom. & Rel. Technol. 9 (7 & 8), 996, 049 8. R.P.W. Scott and C.F. Simpson, J. Chromatogr. 97, 980, 9. Z. Li, S.C. Rutan and S. Dong, Anal. Chem. 68, 996, 4 997 Waters Corporation

rdering Information 5 µm Columns Sentry Guard Column** Dimension. x 50.0 x 50.9 x 50 4.6 x 50 4.6 x 50 Part No. WAT09445 WAT0944 WAT00655 WAT0066 WAT00670 WAT00675 5 µm Cartridge Columns* Dimension.9 x 50.9 x 50 4.6 x 50 4.6 x 50 Part No. WAT09448 WAT00658 WAT00665 WAT0066.5 µm Columns Dimension. x 50 4.6 x 50 4.6 x 75 4.6 x 00 4.6 x 50 Part No. WAT09457 WAT09460 WAT0946 WAT09466 WAT09469.5 µm Cartridge Columns* Dimension 4.6 x 75 4.6 x 00 Part No. WAT0947 WAT09475 5 µm Method Validation Kits (three columns from different batch materials) Dimension. x 50.0 x 50.9 x 50 4.6 x 50 4.6 x 50 Part No. WAT09454 WAT0945 WAT0594 WAT0588 WAT059 5 µm Method Validation Kits (three cartridge columns* from different batch materials) Dimension.9 x 50 4.6 x 50 4.6 x 50 Part No. WAT058 WAT0585 WAT0579.5 µm Method Validation Kits (three columns from different batch materials) Dimension 4.6 x 50 Part No. WAT09478 Description Endfittings Integrated Guard Holder (for Waters steel cartridge columns only) Universal Guard Holder (for any HPLC column) Part No. WAT0755 WAT046905 WAT04690 * Requires reusable endfittings ** Guard columns require the appropriate Sentry Guard Holder. (.9 x 50 mm cartridge columns must use the Universal Guard Holder) For complete Symmetry ordering information, please check box 5 on the business reply card. For your free copy of the Symmetry Applications Notebook, please check box 6 on the business reply card. 997 Waters Corporation

Evaluation of Imitation µbondapak C 8 Columns Waters µbondapak C 8 column was introduced in 97 as the first 0 micron particle size bonded reversed-phase column. Twenty-four years later, it remains one of the most widely used HPLC columns, with applications in many different scientific fields. The selectivity of the µbondapak C 8 column is unique, the result of a carefully controlled combination of hydrophobicity and silanol activity. µbondapak C 8 columns are manufactured only at Waters IS 900-certified Material Synthesis Facility in Taunton, Massachusetts, using proprietary procedures. The popularity of Waters µbondapak C 8 columns has inspired many attempts at imitation. To determine how successful these attempts have been, we recently characterised three imitation columns using a sensitive chromatographic selectivity test. The results are described in this article. Experimental The columns evaluated were all.9 x 00 mm steel columns, except for the 4.6 x 6 mm Imitation C column. The reference column was an authentic Waters µbondapak C 8.9 x 00 mm column. The chromatography was carried out using a Waters 65 LC System, a 77plus Autosampler, a 490 Programmable Multiwavelength Detector, and a Millennium Workstation (all from Waters Corporation). The columns were thermostatted at.4 (± 0.5) C using a Mistral column thermostat. Results and Discussion The columns were first tested for efficiency using acenaphthene as the marker, and a mobile phase of 60/40, v/v, acetonitrile/water flowing at.0 ml/min. The results are summarised in Table. Table : Efficiencies and Tailing Factors for Authentic Waters µbondapak C 8 and Imitation C 8 Columns Column Efficiency USP Tailing µbondapak C 8 0,00.0 Imitation A C 8 0,800 0.9 Imitation B C 8,00 0.9 Imitation Ct C 8,900 0.98 Plates per meter, measured using 5 sigma method Table : Retention Factors and Relative Retentions for Authentic Waters µbondapak and Imitation C 8 Columns The authentic Waters µbondapak C 8 column was found to be the most efficient of the four columns, and also gave the best peak symmetry. The imitation columns were found to have efficiencies that were 5 0% lower, and all showed significant peak fronting (USP Tailing < ). This is an indication that the imitation columns are not as well packed as the authentic Waters µbondapak C 8 column. To evaluate the selectivity of the columns we used a test mixture containing acidic, basic, and non-ionisable compounds (see Figure ). The mobile phase contained methanol (65% v/v) and a ph 70 mm potassium phosphate buffer. Under these conditions, the strong base, propranolol (pk a 9.6) is protonated, and interacts with ionised silanol groups µbondapak Imitation A Imitation B Imitation C Retention Factors Propyl paraben.47.9.7 0.98 Toluene.4.40.90.7 Propranolol 4.5 6.5.6. Acenaphthene 7.60 7.69 6. 5.09 Dibutyl phthalate..4 9.6 7.85 Relative Retention versus Acenaphthene Propyl paraben 0.9 0.67 0.88 0.9 Toluene 0.0 0. 0.05 0.9 Propranolol 0.594 0.85 0.58.57 Dibutyl phthalate.6.48.54.54 through an ion-exchange mechanism. This causes the propranolol peak to tail noticeably. The chromatograms obtained on the four columns are shown in Figure. The retention factors and several relative retentions are summarised in Table. The imitation columns clearly do not match the separation obtained using the authentic Waters µbondapak C 8 column. The largest differences are observed for the strong base, propranolol (marked with arrow). n the authentic Waters µbondapak C 8 column, propranolol elutes midway between toluene and acenaphthene. n the Imitation A C 8 columns, however, propranolol partially coelutes with acenaphthene. 997 Waters Corporation

This indicates that the silanol activity of the alphabond column is higher than that of an authentic Waters µbondapak C 8 column. n the Imitation C C 8 column, the silanol activity is so much higher that propranolol elutes last, as a barely discernable peak. In contrast, on the Imitation B C 8 column propranolol elutes before toluene, indicating reduced silanol activity. Clearly, none of the imitation columns matches the selectivity of an authentic Waters µbondapak C 8 column. Conclusions These results show that there are significant differences between authentic Waters µbondapak C 8 columns and the imitation columns. The imitation columns were found to have lower efficiences, and produced fronting peaks. In addition, we found large differences in selectivity, particularly evident in the retention of basic compounds relative to neutral compounds. This is not surprising, because controlling the retention of basic compounds has been one of the most difficult problems in the synthesis of C 8 silica packings. Research has shown that base retention is affected not only by the bonding processes, but also by many properties of the silica support. In the final analysis, no imitation column matches the performance of an authentic Waters µbondapak C 8 column. Figure : Structures of Test Compounds H C CH Dihydroxyacetone (DHA) C H 7 N H CH CH Acenaphthene C 4 H 9 C 4 H 9 Proplparaben Propranolol Dibutyl phthalate 4 CH Toluene Figure : Chromatograms for Selectivity Test Mixture Separated on Authentic Waters µbondapak C 8 and Imitation Columns Peaks: : DHA : Propyl paraben : Toluene 4: Propranolol 5: Acenaphthene 6: Dibutyl phthalate Peaks: : DHA : Propyl paraben : Toluene 4: Propranolol 5: Acenaphthene 6: Dibutyl phthalate Peaks: : DHA : Propyl paraben : Propranolol 4: Toluene 5: Acenaphthene 6: Dibutyl phthalate 4 5 0 0 0 4 5 0 0 0 5 0 0 0 6 6 6 µbondapak C8 Imitation A C8 Imitation B C8 Peaks: : DHA : Propyl paraben : Toluene 4: Acenaphthene 5: Dibutyl phthalate 6: Propranolol 4 5 0 0 0 6 Imitation C C8 For your copy of the Waters Column Selection Guide for USP Specifications, please check box 7 on the business reply card. 997 Waters Corporation 4

rdering information Particle Pore Column Size Size Dimensions Part No. µbondapak C 8 0 µm 5Å. mm x 00 mm WAT086609.9 mm x 50 mm WAT086684.9 mm x 00 mm WAT074 4.6 mm x 50 mm WAT04470 Bondapak C 8 5-0 µm 5Å.9 mm x 00 mm WAT05875 µbondapak C 8 cartridge column 0 µm 5Å 4.6 mm x 50 mm WAT05860 (requires endfittings) Endfittings (includes sets of reusable endfittings, c-clips and o-rings) WAT0755 How to Reach Us Sales ffices Austria and European Export (Central Europe, CIS, Middle East, India and India Subcontinent Waters Ges.m.b.H. Hietzinger Haupstrasse 45 A-0 Wien Tel: 877 807 Fax: 877 808 Australia Waters Australia Pty. Limited Unit, 8-46 South Street P. Box 84 Rydalmere NSW 6 Australia Tel: 99 777 Fax: 9898 455 Belgium and Luxemburg Waters S.A.-N.V. Raketstraat 60 Rue de la Fusee 60 0 Brussels Tel: 0 76 000 Fax: 0 76 00 Brazil Waters Comercial LTDA Avenida Morumbi 84 - Andar (st floor) CEP. 0470-004 Sao Paulo S.P. Brazil Tel: 0 55 54 7788 Fax: 0 55 50 64 Canada Waters Limited 687 Nashua Drive, Unit 8 Mississauga, ntario L4V V5 Tel: 800 5 475 Fax: 905 678 97 CIS Representation ffice, Moscow U. Miklukho-Miklaia 6/0 787 Moscow Tel: 7 095 6 7000 Fax: 7 095 9 99 Czech Republic Waters Ges.m.b.H hnivcova 4700 Praha 4 Branik Tel: 4 47 867 Fax: 4 47 80 Denmark Waters A/S Baldersbuen 46 640 Hedehusene Tel: 46 598080 Fax: 46 598585 Finland Waters y Kuparitie 00440 Helsinki Tel: 90 506 440 Fax: 90 5064 4 France Waters S.A B.P. 608 78056 St. Quentin en Yvelines Cedex Tel: 048700 Fax: 0487 Germany Waters GmbH Hauptstraße 87 D-65760 Eschborn Tel: 0696 40 06 00 Fax: 0696 48 88 Hong Kong Waters China Ltd. Unit 804, Seaview Commercial Bldg -4 Connaught Road West Sheung Wan, Hong Kong Tel: 85 964 800 Fax: 85 549 680 Hungary Waters kft Vaci ut 0 H-8 Budapest Tel: 6 70 5086 Fax: 6 70 5087 India Waters Pvt. Ltd. No. 6A, II Phase Peenya Industrail Area Bangalore 560080 Tel: 80 894799 Fax: 80 957 Italy Waters S.p.A. Via Achille Grandi, 7 0090 Vimodrone MI Tel: 0 500565 Fax: 0 5087 Japan Nihon Waters K.K. No 5. Koike Bldg. -- Kitashinagawa Shinagawa-ku, Tokyo Tel: 47 79 Fax: 47 76 Malaysia Waters Asia Ltd. 5A Jalan SS5/ Taman Bukit Emas 470 Petaling Jaya Selangor, Malaysia Tel: 704 8600 Fax: 704 8599 Mexico Waters S.A. DE C.V. Adolfo Prieto No. 64 Col. Del Valle 000 Mexico, D.F Tel: 55 54 766 Fax: 55 54 764 The Netherlands Waters Chromatography B.V. Postbus 79 Florijnstraat 9 4870 AJ ETTEN-LEUR Tel: 076 508 700 Fax: 076 508 780 Norway Waters AS Hvamstubben 7 0 Skjetten Tel: 47 68 46 050 Fax: 47 68 46 05 Peoples Republic of China - Beijing Waters China Ltd. Suite 709-7, Xinzhong Building No. Xinzhong Xiejie Gongti Beilu, Dongcheng District Beijing, 0007 P.R. China Tel: 860 0 659 897 Fax: 860 0 6506 044 Poland Waters Sp.zo.o ul. Lektykarska 5 m 0 0-687 Warszawa Tel: 48 44 00 Fax: 48 09 87 Puerto Rico Waters Technologies Corporation P.. Box 6870 Caguas, Puerto Rico 0076-6870 Tel: 787 747 8445, 790 5 Fax: 787 747 8448 Singapore Waters Asia Ltd. (Asia Headquarters) No. Prince Edward Road #0-0 Podium A Bestway Building Singapore 079 Tel: 5 4855 Fax: 5 4655 Spain Waters Chromatografia S.A. Entenza 4- planta baja E-0805 Barcelona Tel: 5 966 Fax: 5 9896 Sweden Waters Sverige AB Box 485, Turebergsvagen S-9 4 Sollentuna Tel: 086 0090 Fax: 086 0095 Switzerland Waters AG Dorfstrasse 0 50 Rupperswil Tel: 06 889 00 Fax: 06 889 059 Taiwan Waters Asia Ltd. Room #60, 6th Floor, No. 6 Sec., Jen-Ai Road Taipei, Taiwan, 068, R..C. Tel: 0 706 8766 Fax: 0 706 9704 UK and Ireland Waters Ltd. The Boulevard Blackmoor Lane Watford, Hertsfordshire WD 8YW Tel: 0 9 86700 Fax: 0 9 90 All ther Countries Waters Corporation 4 Maple Street Milford, MA 0757 USA Tel: 508 478 000 Toll-free: 800 5 475 Fax: 508 87 990 http://www.waters.com/ 5 997 Waters Corporation

Papers to be presented at HPLC 97 Simple and Rugged Solid-Phase Extraction (SPE) Method for the Determination of Tetracyclines in Serum by HPLC Using a Volatile Mobile Phase Yung-Fong Cheng, Dorothy J. Phillips and Uwe D. Neue Waters Corporation, 4 Maple Street, Milford, MA 0757 USA Due to its high sensitivity, high selectivity and high sample throughput, LC/MS/MS has become the choice for the analysis of drugs and their metabolites in biological fluids such as serum and urine. This paper reports a simple and reproducible SPE method for the determination of tetracycline (TC), minocycline (MC) and demeclocycline (DCC) in porcine serum using a volatile HPLC mobile phase. High ph Stability of Silica-Based Reversed-Phase HPLC Packings Tom Walter, Bonnie Alden and Ray Fisk Waters Corporation, 4 Maple Street, Milford, MA 0757 USA Recently there has been considerable interest in the stability of silica-based reversed-phase packings when exposed to high ph mobile phases. The most common failure mode is loss of efficiency and peak symmetry due to voiding, as a result of dissolution of the silica particles. In contrast to the results reported by other researchers, we find no correlation between stability and the method of preparation of the silica particles Applications of a Polymeric Extraction Sorbent for Isolating Highly Polar Drugs From Biological Fluids Dorothy J. Phillips, Edouard S. P. Bouvier, Mark Capparella, Yung-Fong Cheng, Pamela Iraneta, Uwe D. Neue and Laura L. Bean Waters Corporation, 4 Maple Street, Milford, MA 0757 USA Method development experts in the pharmaceutical industry are striving to streamline sample preparation. Considerable time and effort are consumed in choosing the appropriate solid-phase extraction (SPE) sorbent and extraction protocol. The new Waters asis HLB extraction cartridges address the problem of low recovery of polar analytes. This paper includes the extraction procedures, HPLC methods and the results for several highly polar drugs. Development of Methods for Extraction of Neutral Steroids From Biological Matrices With a Polymeric Solid-Phase Sorbent Dorothy J. Phillips, Mark Capparella, Yung- Fong Cheng, Pamela Iraneta, Uwe D. Neue Waters Corporation, 4 Maple Street, Milford, MA 0757 USA Steroids have many biological functions, making their analysis important in biomedical applications. The ratio of the amount of a steroid to that of extraneous material is very low in biological fluids. Therefore, extraction of the steroid from the biological matrix is necessary prior to HPLC analysis. The solid-phase extraction method development process, giving details at each step from sample preparation to elution, will be presented. Simple and Fast Method Development For HPLC and Sample Preparation Yung-Fong Cheng, Dorothy J. Phillips, Uwe D. Neue and Laura Bean Waters Corporation, 4 Maple Street, Milford, MA 0757 USA The development of assays for drugs and their metabolites from serum is time-consuming since optimisation of the HPLC method and the sample preparation is difficult and tedious. It would be desirable to have a well thought out strategy for HPLC method development which can provide good peak shapes and good resolution for a wide range of compounds. In this paper, a carefully planned HPLC method development protocol using Waters Symmetry columns for the separation of chlordiazepoxide and its metabolites (ranging from basic to uacidic in nature), will be presented. 997 Waters Corporation 6