ACQUITY UPLC System. Application Notebook December 2005

Transcription

1

2

3

4 4 ACQUITY UPLC SYSTEM DECEMBER 25 ACQUITY UPLC System Application Notebook December Recent Developments in Columns for UPLC Eric S. Grumbach, Thomas E. Wheat, and Jeffrey R. Mazzeo Transfer of the USP Human Insulin Related Compounds HPLC Method to the ACQUITY UPLC System Tanya L. Jenkins and Patricia McConville Validation of a UPLC Method for a Benzocaine, Butamben, and Tetracaine Hydrochloride Topical Solution Andrew J. Aubin and Tanya L. Jenkins Meeting Analytical Challenges with UPLC MS John Van Antwerp, Richard DePinto, and Dale Jansen Enabling Significant Improvements for Peptide Mapping with UPLC Jeffrey R. Mazzeo, Thomas E. Wheat, Beth L. Gillece-Castro, and Ziling Lu Analysis of Prostaglandins with the Quattro Premier XE Kate Yu and Donald Kwet

5

6 6 ACQUITY UPLC SYSTEM DECEMBER 25 Recent Developments in Columns for UPLC Eric S. Grumbach, Thomas E. Wheat, and Jeffrey R. Mazzeo Waters Corporation, Milford, Massachusetts Additional UPLC chemistries provide flexibility for methods development. Ultra Performance LC (UPLC ) enables chromatographers to meet the challenges of developing separations that completely characterize the constituents of samples by providing significant improvements in resolution, speed, and sensitivity. These improvements are achieved through the use of columns with 1.7 m particle packings and a matching family of low-dispersion, high-speed instrumentation developed simultaneously to provide full compatibility between chemistry and instrumentation. Recently, additional bonded phases have been developed to focus on improving resolution by manipulating selectivity. A 1.7 m particle-packed column provides significant improvements in resolution due to higher efficiencies. Separation sample components, however, still requires a bonded phase that provides both retention and selectivity. Four bonded phases are available for UPLC separations: Waters ACQUITY UPLC Bridged Ethyl-siloxane/silica Hybrid (BEH) C 18 and C 8 (straight chain alkyl columns), ACQUITY UPLC BEH Shield RP 18 (embedded polar group column), and ACQUITY UPLC BEH Phenyl (phenyl group tethered to the silyl functionality with a C 6 alkyl). Each provides a different combination of hydrophobicity, silanol activity, hydrolytic stability, and chemical interaction with the analytes. Experimental Conditions Columns: ACQUITY UPLC BEH C 18, C 8, Shield RP 18, Phenyl Column Dimensions: mm, 1.7 m Mobile Phase A: Water Mobile Phase B: Methanol Isocratic Conditions: 72 A; 28 B Flow Rate:.5 ml/min Injection Volume: 5. L Weak Needle Wash: 28 methanol Sample: Nitroaromatics Sample Diluent: 3 methanol Sample Concentration: 1 g/ml Temperature: 5 C Detection: 254 nm Sampling Rate: 2 pts/s Time Constant:.1 Instrument: Waters ACQUITY UPLC System with PDA Detector Results The analysis of nitroaromatics from EPA Method 833 is often performed on multiple stationary phases to resolve two sets of critical pairs. The high efficiency of UPLC columns provides the baseline separation of 14 nitroaromatics on the two linear alkane bonded phases (Figure 1). Compared to the C 18 column, the shorter chain length C 8 bonded phase is less hydrophobic, and, therefore, less retentive. Although selectivity differences seldom result from chain length differences, a change in peak elution has occurred. Embedded polar group columns often proved a selectivity that is unique compared to linear alkanes. The ACQUITY UPLC BEH Shield RP 18 column includes an embedded carbamate group that shows preferential retention of hydrogen-bond donors. Additional attributes include low silanol activity and aqueous compatibility. Columns with phenyl ligands provide yet another alternate selectivity. Due to the bonding orbital interactions, phenyl columns provide unique and specific selectivity with aromatic compounds and other analytes with similar electrons. Conclusions Stability over a broad ph operating range (ph 1 12) combined with the several available bonded phases, provides flexibility for methods development. This flexibility enables methods development to be more efficient, allowing products to be brought to market faster. The power of these ultra-efficient columns is combined with a low-dispersion Ultra Performance LC system to successfully transfer existing HPLC methods or to develop new, fast chromatographic methods that offer substantial improvements in resolution, sensitivity, and sample throughput.

7 DECEMBER 25 ACQUITY UPLC SYSTEM 7 Figure 1: Multiple ACQUITY UPLC bonded phases with different selectivity provide flexibility for methods development. Analytes: (1) HMX; (2) RDX; (3) 1,3,5-TNB; (4) 1,3-DNB; (5) NB; (6) Tetryl; (7) TNT; (8) 2-Am-4,6-DNT; (9) 4-Am-2,6-DNT; (1) 2,4-DNT; (11) 2,6-DNT; (12) 2-NT; (13) 4-NT; (14) 3-NT.

8 8 ACQUITY UPLC SYSTEM DECEMBER 25 Transfer of the USP Human Insulin Related Compounds HPLC Method to the ACQUITY UPLC System Tanya L. Jenkins and Patricia McConville Waters Corporation, Milford, Massachusetts The process of method transfer can be tedious and time-consuming, especially for a validated method. As a result, many LC methods developed using dated technology are still in use today. However, with the incentive of significant potential improvements in chromatographic separation speed and resolution, taking the necessary steps to transfer an existing protocol over to newer, better performing technology becomes less daunting. The transfer of the USP (United States Pharmacopeia) method for the related compounds of human insulin is particularly challenging, as it often requires re-optimization for each analysis. In this application note, we outline the successful method transfer steps from the USP-recommended mm, 5 m L1 column run on a traditional HPLC system to a Waters ACQUITY UPLC BEH mm, 1.7 m column on a Waters ACQUITY Ultra Performance LC System. To optimize the new method, experimental van Deemter curves were generated to determine the optimal flow rate. Due to the high molecular weight of the human insulin molecule ( 58), diffusion into the pores of the packing material is slower. Therefore, the optimal flow rate for the separation is relatively slow and its impact on run time was considered for the final method. Instrument considerations, such as detection parameters for sensitivity and injector carryover performance, were optimized to maximize the benefits of the ACQUITY UPLC system for this method. The resulting UPLC method reduced the overall run time by 6, with improved resolution. The data meets or exceeds acceptance criteria. Human insulin is a relatively small protein containing 51 amino acids in two polypeptide chains. The drug product is a biopharmaceutical synthesized through recombinant DNA technology. The insulin made by these cells is identical to the insulin made by the human pancreas. The USP method for the related compounds of human insulin is a 68-min separation involving the isocratic elution of the main component (within a specified retention window of min) and the major related compound (A21 desamido insulin), followed by the gradient elution of the high MW impurities (1). The run is followed by a re-equilibration step which is typically 23 min (5 column and 3 system volume). The elution time of the main insulin peak is extremely sensitive to the amount of organic modifier in the mobile phase. Adjustments to the gradient table are necessary for each analysis to ensure that the main insulin peak is eluted within the specified retention window to achieve sufficient resolution with A21 desamido insulin peak. A change of less than.5 of the organic modifier can cause the peak to elute outside of this retention window, making the on-line mixing and gradient performance of the instrumentation critical for this application. The method also utilizes high salt concentrations at low ph. Small changes in either of these parameters impact peak shape and selectivity. Elution times are also sensitive to changes in temperature. A difference of 1 2 C can push the retention of the main insulin peak outside of the specified window. Injector carryover is also an important consideration. Sample concentrations of up to 4 mg/ml are used to measure impurities and managing carryover can be challenging for HPLC injectors. A method is desired which will reduce the run time. Time is crucial since samples and standards are only useable for up to 12 h. The current USP method with a total analysis time of 91 min is long, considering the optimization time required before samples can be analyzed. However, any reduction of analysis time cannot sacrifice the performance of the method. Precision and carryover must still meet requirements. Method Scaling Equations The HPLC to UPLC method transfer process can be streamlined by using a series of equations to geometrically scale the original method to the new column dimensions (2). These equations account for changes in gradient times, flow rate, and injection volume. They do not compensate for changes in system volume, column selectivity or column load (mass or volume). To start, the gradient steps are scaled from the HPLC column to the UPLC column:

9 DECEMBER 25 ACQUITY UPLC SYSTEM 9 Figure 1: The ACQUITY UPLC System Console Calculator. Geometrically Scaled Gradient Time = UPLC Column Length HPLC Column Length Next, the flow rate is scaled taking into account the difference in the internal diameters of the columns: Geometrically Scaled Flow Rate = (UPLC Column Diameter) (HPLC Column Diameter) 2 HPLC Gradient Time 2 HPLC Flow Rate The flow rate should then be further scaled to take into account the new optimal linear velocity of the separation using the smaller 1.7 m particles. For 2.1 mm i.d. columns, appropriate starting points are typically 65 L/min for small molecules and 1 L/min for high MW compounds. To keep the column volumes proportional, the gradient steps should be readjusted for the new flow rate: UPLC Gradient Time = Scaled Flow Rate x Gradient Time UPLC Flow Rate The injection volume is scaled taking into account the volume of the columns: UPLC Injection Volume = UPLC Column Volume HPLC Column Volume HPLC Injection Volume Alternatively, the ACQUITY UPLC System Console Calculator (Figure 1) will scale the method automatically using the same scaling principles outlined here.

10 1 ACQUITY UPLC SYSTEM DECEMBER 25 Absorbance (AU) Insulin A Time (min) Figure 2: Chromatogram of the 68-min USP HPLC method for the human insulin related compounds assay. Original USP HPLC Method System: Alliance 2695 XC Separations Module; 2996 Photodiode Array Detector; Empower Software Column: Symmetry 3 C 18, 4.6 mm 25 mm, 5 m Detection: 214 nm, 1.2 nm digital bandwidth, 1 pt/s, 1. s time constant Eluents: Buffer:.2 M Sodium Sulphate, ph 2.3; A: 82/18 Buffer/Acetonitrile; B: 5/5 Buffer/Acetonitrile Gradient: 36 min isocratic at 24.5 B min linear gradient to 64 B min isocratic at 64 B min linear gradient to 24.5 B RT Window: min Flow Rate: 1 ml/min Temp: 35 C Sample: USP Human Insulin Reference Standard; 3.75 mg/ml in.1 M Hydrochloric Acid Volume: 2 L Wash: Extended Wash Cycle with 6:3:1 of.1 Phosphoric Acid: Acetonitrile: Isopropyl Alcohol Final Transferred UPLC Method System: ACQUITY UPLC System with Photodiode Array Detector; Empower Software Column: ACQUITY UPLC BEH C 18, 2.1 mm 1 mm, 1.7 m Detection: 214 nm, 12. nm digital bandwidth, 5 pts/s,.3 s time constant Eluents: Buffer:.2 M Sodium Sulphate, ph 2.3; A: 82/18 Buffer/Acetonitrile B: 5/5 Buffer/Acetonitrile Gradient: 14.4 min isocratic at 26 B min linear gradient to 64 B min isocratic at 64 B min linear gradient to 26 B RT Window: 6 1 min Flow Rate: 28 L/min Temp: 35 C Sample: USP Human Insulin Reference Standard; 1.25 mg/ml in.1 M Hydrochloric Acid Volume: 1.8 L characterized full loop Strong Wash: 2 L of 6:3:1 of.1 Phosphoric Acid: Acetonitrile: Isopropyl Alcohol Weak Wash: 12 L of.1 M Hydrochloric Acid

11 DECEMBER 25 ACQUITY UPLC SYSTEM Insulin Absorbance (AU) A Time (min) Figure 3: Chromatogram of the 27-min UPLC method for the human insulin related compounds assay. 25, Number of theoretical plates per meter 2, 15, 1, 5, Large molecule MW = 588 Small molecule MW = Flow rate (µl/min) Figure 4: Dependence of the number of theoretical plates on the flow rate.

12 12 ACQUITY UPLC SYSTEM DECEMBER Absorbance (AU) High concentration standard Absorbance (AU) Blank Time (min) Time (min) Figure 5: Carryover of human insulin on the ACQUITY UPLC system. Results and Discussion The USP method was scaled geometrically and then optimized, according to the USP specifications, to have the main insulin peak elute within the required retention window. This process involved increasing the organic modifier composition by 1.5 for the isocratic portion of the separation. The scaled injection volume and sample concentration were adjusted such that the height of the main insulin peak was within the linear range of the detector. The resulting UPLC method had a run time of 27.2 min (a 6 time reduction compared to the HPLC method), with a total analysis time of 37.5 min (compared to the 91-min HPLC analysis a time reduction of 54 min). To determine the optimal flow rate for the UPLC column, a plot of the number of theoretical plates versus flow rate was generated (Figure 4). For small molecules, the optimal flow rate for a 2.1 mm i.d. column packed with 1.7 m particles is typically around 65 L/min. However, for larger molecules the optimal flow rate is lower, and for the human insulin method it was under 1 L/min. Since the goal of this method transfer is to improve run time, the final method was chosen to operate above the optimal flow rate. Even when operating above the optimum flow rate for the separation, improvements in resolution were observed. The relative peak widths for the UPLC separation (Figure 3) were narrower than those of the orginal HPLC method (Figure 2). Improved resolution was attained with an impurity in the tail of the main insulin peak, as well as with some of the other lower level impurities. The HPLC method had resolution between the main insulin peak and the A21 desamido insulin peak of 5., while the UPLC method had a resolution of 6.8. Signal-to-noise for the A21 desamido insulin peak was 1:1. The narrow peaks generated by the UPLC separation required the detection parameters to be re-optimized. The data rate was increased and the filtering constant decreased to be more compatible with the chromatography and to maximize sensitivity. The performance of the human insulin method on the ACQUITY UPLC system was excellent (Table I), with precision values far below typical requirements. Retention time reproducibility was under.5 at.23. The area reproducibility of the main insulin peak was below 1 at.21 and the A21 desamido insulin peak was well below 1 at only 1.6. The dual wash capabilities of the ACQUITY UPLC system efficiently removes residual sample from the system, resulting in negligible carryover. The wash cycle does not contribute to injection cycle time as it occurs post-injection, while the sample is running. To measure carryover, a set of high concentration standards (1 mg/ml) was used. The injection sequence was 2 blanks, 3 standards at.5 (carryover specification), 6 high concentration standards, and 3 blanks. Table II shows the first injection after the blank had a carryover of.4 and by the third blank injection, no insulin could be detected. Figure 5 shows the high concentration standard compared to the first blank injection.

13 DECEMBER 25 ACQUITY UPLC SYSTEM 13 Table I: Reproducibility values for the human insulin UPLC method. Peak Retention Time RSD Peak Area RSD Human Insulin A21 Desamido Insulin Table II: Carryover of human insulin in subsequent blank injections. Injection Average Peak Height mau Carryover Standard Blank Blank Blank 3 none detected none detected Conclusions We have shown that challenging USP HPLC methods can be successfully transferred to the Waters ACQUITY UPLC system, with numerous chromatographic performance benefits. Transfer of the human insulin method was accomplished by simple scaling with an adjustment to the sample concentration and injection volume. The gradient table was adjusted for the new method according to the USP guidelines. The new UPLC method offered a 6 reduction in run time over the USP HPLC method (a 54- min reduction in total analysis time) with improved resolution. UPLC reproducibility for both peak area and retention time was very good, especially for such a chromatographically sensitive sample. In addition, sample carryover was easily managed and minimized by the ACQUITY UPLC system. This exercise clearly demonstrates the advantages that can be realized by transferring from HPLC to UPLC methodology. References (1) The United States Pharmacopeia USP28, the National Formulary NF23 (United States Pharmacopeial Convention, Inc., 24), pp (2) E. Grumbach, T. Wheat, and J. Mazzeo, An Efficient HPLC to UPLC Method Transfer Protocol, poster presented at HPLC 25, June 25, Stockholm, Sweden (Waters Literature Code WA41923).

14 14 ACQUITY UPLC SYSTEM DECEMBER 25 Validation of a UPLC Method for a Benzocaine, Butamben, and Tetracaine Hydrochloride Topical Solution Andrew J. Aubin and Tanya L. Jenkins Waters Corporation, Milford, Massachusetts Benzocaine (4-Aminobenzoic acid ethyl ester), Butamben (Butyl 4-aminobenzoate), and Tetracaine (4 (Butylamino)benzoic acid 2-(dimethylamino)ethyl ester) are topical anesthetics. The formulated mixture of these three active ingredients is indicated for the production of anesthesia of all accessible mucous membranes except the eyes. In this application note, we demonstrate the successful development and validation of an Ultra Performance LC (UPLC ) method for the analysis of these compounds. Experimental Materials Benzocaine, butamben, tetracaine HCl, benzalkonium chloride, cetyl dimethyl ethyl ammonium bromide, ammonium bicarbonate, and ammonium hydroxide were purchased from Sigma-Aldrich Co. (St Louis, Missouri). Water was purified with a Milli-Q system (Millipore, Billerica, Massachusetts). Acetonitrile was purchased from Mallinckrodt Baker, Inc. (Phillipsburg, New Jersey) and was of HPLC grade. The formulated topical solution was purchased from a local pharmaceutical supplier and was labeled to contain 14. benzocaine and 2. each of butamben and tetracaine HCl as well as the inactive ingredients benzalkonium chloride (.5) and cetyl dimethyl ethyl ammonium bromide (.5). All bottles of the topical solutions were of the same production lot. UPLC Apparatus and Conditions The Waters ACQUITY UPLC System consisted of a Binary Solvent Manager, Sample Manager, and an ACQUITY UPLC Tunable UV (TUV) Detector. System control, data collection, and data processing were accomplished using Waters Empower 2 Chromatography Data Software. Separations were performed at 4 C using an ACQUITY UPLC Bridged Ethyl-siloxane/silica Hybrid (BEH) C m, 2.1 mm 5 mm column. Elution Absorbance (AU) Benzocaine.29 min Butamben.65 min Tetracaine 1.5 min Time (min) Figure 1: UPLC separation of Benzocaine, Butamben, and Tetracaine.

15 DECEMBER 25 ACQUITY UPLC SYSTEM 15 Table I: Detector timetable. Time range (min) Wavelength (nm) Compound RT (min) Benzocaine Butamben Tetracaine 1.5 Table II: Working standard solutions. Standard Benzocaine Stock (ml) Butamben Tetracaine HCl Stock (ml) Approximate concentrations of working solutions (mg/ml) Benzocaine Butamben Tetracaine HCl Level Level Level Level Level x min Intensity Intensity Intensity min 2.x min 3.x m/z Figure 2: Mass spectra of benzocaine (top), butamben (middle), and tetracaine (bottom).

16 16 ACQUITY UPLC SYSTEM DECEMBER 25 was accomplished using a 1. mm ammonium bicarbonate mobile phase (adjusted to ph 1. with ammonium hydroxide) filtered through at.45 m membrane, and acetonitrile (6:4) at 1. ml/min., one microliter injections were used for both standards and samples. The sample manager was equipped with a 5 L loop and used the Partial Loop Needle Overfill mode with 1 L air gaps. To minimize carryover, 12 L of the weak wash (6:4 water/acetonitrile) and 4 L of the strong wash (1:9 water/acetonitrile) were used. Detection wavelengths were optimized for each compound and collected on a timed event basis (Table I). A sampling rate of 2 points/s and a filter constant of.1 s were used throughout. The total run time was 1.5 min (Figure 1). Preparation of Standard Solutions Stock Solutions A standard solution (2. ml) of benzocaine at 1. mg/ml and a standard solution (2. ml) containing.15 mg/ml each of butamben and tetracaine hydrochloride was prepared by dissolving appropriate amounts of each in the diluent (a mixture of 1. mm Table III: Specificity results. Name RT (min) Resolution Purity Angle Purity Threshold Unknown Benzocaine Unknown Butamben Tetracaine Table IV: Linearity results. Name R^2 Y Intercept Difference Equation Benzocaine Y 2.49e 6X 5.47e 3 Butamben Y 4.71e 6 X 5.39e 2 Tetracaine Y 4.24e 6X 2.9e 2 91, 819, 728, 637, 546, Area 455, 364, 273, 182, 91, Amount Figure 3: Calibration curve for benzocaine.

17 DECEMBER 25 ACQUITY UPLC SYSTEM ,. 192,. 168,. Butamben 144,. 12,. Area 96,. Tetracaine 72,. 48,. 24, Amount Figure 4: Calibration curves for butamben and tetracaine. Table V: Repeatability at the 5 level (n 6). RT (min) Benzocaine Butamben Tetracaine Mean Std. Dev RSD Amount Mean Std. Dev RSD Table VI: Repeatability at the 1 level (n 6). RT (min) Benzocaine Butamben Tetracaine Mean Std. Dev RSD Amount Mean Std. Dev RSD

18 18 ACQUITY UPLC SYSTEM DECEMBER Residual deviation () Standard level Figure 5: Residuals plot for standard curves of benzocaine ( ), butamben ( ), and tetracaine ( ). Table VII: Repeatability at the 15 level (n 6). RT (min) Benzocaine Butamben Tetracaine Mean Std. Dev RSD Amount Mean Std. Dev RSD ammonium bicarbonate at ph 1. and acetonitrile (6:4)). Working Solutions Working standard solutions (5 levels, 1. ml of each) were prepared by diluting appropriate amounts of stock solutions in the diluent to give concentrations ranging from.1.3 mg/ml for benzocaine and mg/ml for butamben and tetracaine hydrochloride (Table II). Topical Solution Sample Preparation About 3 mg of Topical Solution was accurately weighed and transferred to a 2 ml volumetric flask. Approximately 18 ml of diluent was added and mixed on a wrist action shaker for 5 min. The sample was diluted to final volume and mixed thoroughly. An aliquot of the sample was transferred to a 2-mL vial and capped. Chromatographic Analysis Procedure Equal volumes (1. L) of the 5 standard preparations and the topical solution sample preparation were separately injected into the chromatograph, chromatograms recorded, peak areas were measured for the 3 major peaks. A calibration curve was then created based on the 5 standard preparations. The amounts were calculated, in mg/ml, for benzocaine, butamben and tetracaine hydrochloride in the portion of topical solution sample preparation by comparing the area of each peak to the linear regression line of the calibration curve, based on the five standard preparations. The amount was calculated, by weight of benzocaine, butamben, and tetracaine hydrochloride in the topical solution using the formula: Concentration (mg/ml) (2 ml Weight Topical Solution in Volumetric (mg)) 1

19 DECEMBER 25 ACQUITY UPLC SYSTEM 19 The current monograph from the USP states that Benzocaine (14), Butamben (2), and Tetracaine Hydrochloride (2) Topical Solution should contain not less than 9. and not more than 11. of the labeled amounts of benzocaine, butamben, and tetracaine hydrochloride. Suitability Criteria Based on the chromatograms of standard preparations, the retention times (in minutes) are about.3 for benzocaine,.65 for butamben, and 1.5 for tetracaine; the resolution, R, between the benzocaine peak and the butamben peak and between the butamben peak and the tetracaine peak is not less than 8; and the relative standard deviation for retention time of replicate injections is not more than 1. for each of the 3 analyte peaks. The relative standard deviation for amount of replicate injections is not more than 2. for each of the 3 analyte peaks. Assay Validation and Results Specificity To demonstrate specificity, injections of individual reference standard, a diluent blank, and a solution that contains approximate concentration of labeled nonactive ingredients were evaluated. The reference standards and diluent blanks were prepared using the same protocols as the assay preparation to ensure no contamination is added during the sample preparation steps. Samples were evaluated using photodiode array detection and MS detection and peak purity were assessed using Empower 2 software. Acceptance Criteria for Specificity The diluent blanks must show no interference with the peaks of the active ingredients. Resolution between the main sample peaks should be greater than or equal to 2.. The resolution factors between all sample peaks and any other peak should be greater than or equal to 2.. Any co-elution of significant nonactive ingredient, excipient or unknown peaks must be clearly noted in the final reporting of the results. To determine peak purity, the threshold must exceed the peak purity angle for the main sample peaks. Table VIII: Intermediate precision results. Active Benzocaine Butamben Tetracaine Analyst 1 Analyst 2 Diff Analyst 1 Analyst 2 Diff Analyst 1 Analyst 2 Diff Mean Std. Dev RSD Table IX: Reproducibility results. Active Benzocaine Butamben Tetracaine Lab 1 Lab 2 Diff Lab 1 Lab 2 Diff Lab 1 Lab 2 Diff Mean Std. Dev RSD Table X: Tested robustness criteria. Parameter Specified Conditions Modified Conditions Wavelength (nm) 22, 29, , 295, 312, and 215, 285, 32 Flow Rate (ml/min) 1..9,.95, 1.5, 1.1 Column Temperature ( C) 4 38, 39, 41, 42 Injection Volume ( L) 1..8,.9, 1.1, 1.2 Mobile Phase Composition 6/4 5/5, 55/45, 65/35, 7/3 Buffer Concentration mmol 1 8, 9, 11, 12 Buffer ph 1. 9., 9.5, 1.5, 11. Sample Prep Shake Time (min) 5 2, 5, 1

20 2 ACQUITY UPLC SYSTEM DECEMBER 25 Table XI: Method robustness results. Method Condition Benzocaine Butamben Tetracaine Wavelengths 5 nm *Specified Wavelengths Wavelengths 5 nm Flow Rate.9 ml/min Flow Rate.95 ml/min *Flow Rate 1. ml/min Flow Rate 1.5 ml/min Flow Rate 1.1 ml/min Column Temperature 38 C Column Temperature 39 C *Column Temperature 4 C Column Temperature 41 C Column Temperature 42 C Injection Volume.8 L Injection Volume.9 L *Injection Volume 1. L Injection Volume 1.1 L Injection Volume 1.2 L Mobile Phase Composition 5/ Mobile Phase Composition 55/ *Mobile Phase Composition 6/ Mobile Phase Composition 65/ Mobile Phase Composition 7/ Buffer Concentration 8 mmol Buffer Concentration 9 mmol *Buffer Concentration 1 mmol Buffer Concentration 11 mmol Buffer Concentration 12 mmol Buffer ph Buffer ph *Buffer ph Buffer ph Buffer ph Sample Prep Shake Time 2 min *Sample Prep Shake Time 5 min Sample Prep Shake Time 1 min * Conditions as specified in the method. 1 Variation of 2. compared to the result obtained at the specified method conditions.

21 DECEMBER 25 ACQUITY UPLC SYSTEM 21 Table XII: Recovery of spiked assay samples at 3 levels (n 6). Benzocaine Butamben Tetracaine Spiked at 8 of label Spiked at 1 of label Spiked at 12 of label Specificity Results Injections of a diluent blank showed no extraneous peaks. Injections of individual reference standards gave the following retention times: benzocaine.29 min, butamben.65 min, and tetracaine 1.5 min. No other peaks were noted in the chromatograms from the individual standards. A solution that contained the inactive ingredients benzalkonium chloride (.5) and cetyl dimethyl ethyl ammonium bromide (.5) in water was analyzed. No peaks were noted in the regions of the active ingredients. Photodiode array analysis and single quadrupole mass spectrometry detection were applied to a sample. Resolution requirements outlined in the acceptance criteria for specificity were easily met (Table III). Photodiode array peak purity results of the sample showed purity angles less than purity thresholds for the 3 peaks of interest, indicating each peak had a high degree of peak homogeneity. Evaluation of the mass spectral data confirmed spectral identity of the 3 peaks of interest (Figure 2). Based on these results, the method was determined to be specific for the analysis of benzocaine, butamben, and tetracaine. Linearity Serial dilutions from a stock standard solution were made to obtain 5 linearity solutions incorporating levels from 5 to 15 of the nominal analytical concentration of the sample components. Each solution was injected in duplicate. The linear regression of these injections provided the correlation coefficient, y-intercept, and the residual sum of squares. Acceptance Criteria for Linearity The correlation coefficient should not be less than.999 for all the sample components and the y-intercept should be within 2. when compared to the 1 level. The plot of the residuals should indicate linearity. Linearity Results The linearity of all 3 compounds met the criteria described above (Table IV). Linearity plots are shown in Figures 3 and 4. Residuals at all 5 standard levels were all less than 2 and indicated linearity (Figure 5). Precision Repeatability The repeatability of the main sample peaks was measured by making 6 replicate injections of a single standard solution at each of 3 levels (5, 1, and 15). Acceptance Criteria for Precision Repeatability The RSD for amount should not be more than 2. for each of the main sample components at each level. The RSD for retention time should not be more than 1. for each of the main sample components at each level. Precision Repeatability Results The repeatability for all three compounds met the acceptance criteria (Tables V VII). Precision Intermediate Precision To establish the effects of random events on the precision of the analytical procedure, 2 analysts prepared and analyzed 6 sample assay preparations from 1 batch and 2 preparations each from 2 additional batches. Each analyst prepared their own standards and solutions, used a column from a different lot, and used different systems to evaluate the sample solutions. Acceptance Criteria for Precision-Intermediate Precision The RSD obtained for the 1 preparations for active ingredient by both analysts should not be more than 2.. The mean analyst 1 result for each batch tested should not differ from the mean analyst 2 result for sample peaks by more than 2.. Precision Intermediate Precision Results Intermediate precision results for both analysts met the criteria described above, demonstrating acceptable intermediate precision for the assay. Results are summarized in Table VIII. Precision Reproducibility Reproducibility is assessed by means of an inter-laboratory trial. Analysts from 2 different labs (different from the analysts involved in the intermediate precision) prepared and analyzed 6 sample assay preparations from one batch and 2 preparations each from 2 additional batches. Each analyst prepared their own standards, solutions, used a column from a different lot, and different systems to evaluate the sample solutions. Acceptance Criteria for Precision-Reproducibility The RSD obtained for the 6 preparations by both analysts should not be more than 2.. The mean lab 1 result for each batch tested should not differ from the mean lab 2 result for the sample peaks by more than 2.. Precision Reproducibility Results Precision reproducibility results from both laboratories met the criteria described above demonstrating acceptable reproducibility for the assay. Results are summarized in Table IX.

22 22 ACQUITY UPLC SYSTEM DECEMBER 25 Robustness Robustness is the ability of a method to remain unaffected by small changes in conditions. If the changes are within the limits that produce acceptable chromatography, the method is considered robust. Six replicate injections of a standard and a sample solution were performed under both the specified and modified conditions shown in Table X. Acceptance Criteria For Robustness The average values obtained at the modified conditions should not differ by more than 2. for the sample components from those values obtained using the specified conditions. Robustness Results The results for the various method modifications are summarized in Table XI. For changes applied to the method, calculated amounts of the active ingredients did not vary by more that 2 (relative to the specified method conditions), except in the case of column temperature, where the results from the 38 C column temperature fell outside of this range for all 3 active ingredients (Table XI). These results demonstrate that the method was robust, although care must be taken to ensure the specified column temperature. Accuracy/Recovery The 3 analytes were spiked into a blank sample matrix at 3 levels: 8 of label, 1 of label, and 12 of label. These spiked samples were prepared according to topical solution sample preparation directions and analyzed according to the chromatographic analysis procedure. Amounts were determined using the same quantitation procedure as was used in the final method procedure. The percent recovery was then calculated. Acceptance Criteria For Accuracy/Recovery The measured value of the sample peaks in the spiked placebos should be within 6 2. of the spiked value. Accuracy/Recovery Results The percent recovery values are summarized in Table XII. Recovery values for all 3 compounds at the 3 spike levels were within 2 of the expected value. Conclusions A linear, accurate, selective, robust, and reproducible UPLC method for the determination of Benzocaine, Butamben, and Tetracaine Hydrochloride Topical Solution was successfully validated. By taking advantage of ACQUITY UPLC system performance and BEH C m columns, the entire method validation process was shortened from weeks to days, yielding a rugged method with superior resolution and speed. Acknowledgments The technical assistance of Katherine Hynes and the participation of Mark Benvenuti and Michael D. Jones are gratefully acknowledged.

23 DECEMBER 25 ACQUITY UPLC SYSTEM 23 Meeting Analytical Challenges with UPLC MS John Van Antwerp, Richard DePinto, and Dale Jansen Waters Corporation, Milford, Massachusetts The utilization of single quadrupole mass spectrometers for drug discovery and development has increased significantly over the past five years, particularly for Open Access walk-up and Mass-Directed Autopurification environments. The ongoing demand for higher throughput and increased speed, sensitivity, and resolution continues to drive chemists to seek out the latest in high performance technologies. Recently, with the introduction of the Waters ACQUITY Ultra Performance LC System, many chemists are considering the addition of this breakthrough chromatographic technology to further improve their overall data quality and productivity. To demonstrate, we have utilized the ACQUITY UPLC System with a Waters ZQ 2 Mass Detector for the analysis of a representative selection of pharmaceutical compounds. The results address a common concern when peaks are very narrow (as with UPLC), and the resulting MS spectral quality from such fast analyses. In this applications note, we show the outstanding performance of the ZQ 2 with narrow peaks in both an ESI mode (Electrospray Ionization) and in an ESCi multi-mode ionization (both ESI and APCI) in the analysis of a multi-component drug test mixture (Figure 1). Objectives When comparing UPLC MS to traditional HPLC MS analysis, the following high performance benefits can be expected: Higher sample throughput Enhanced qualitative detection and sensitivity Increased selectivity and resolution More quality information obtained in less time Added capabilities from a single benchtop system Improved overall efficiency and productivity Method 1 (See Figures 1 2) LC Conditions LC System: ACQUITY UPLC System Column: ACQUITY UPLC BEH C 18, 2.1 mm 5 mm, 1.7 m Flow Rate:.75 ml/min Intensity 1.1x1 8 1.x1 8 9.x1 7 8.x1 7 7.x1 7 6.x1 7 5.x1 7 4.x1 7 3.x1 7 1.x1 7 1.x1 7 Ephedrine Caffeine Doxylamine Lidocaine -.62 Acetanilide Propraolol Diphenhydramine -.86 Bromazapam Trimipramine Oxybutynin Terfenadine Diazapam Time (min) Figure 1: UPLC MS enables accelerated, high-resolution separations with second typical peak widths.

24 24 ACQUITY UPLC SYSTEM DECEMBER Terfendine Trimipramine Propranolol Lidocaine Caffeine Figure 2: Individual spectra for five compounds in the test mix, where [M H] ions are easily discernable. Compound name: terfenadine Correlation coefficient r = , r 2 = Calibration curve: * x Response type: External Std, Area Curve type: Linear, Origin: Exclude, Weighing" 1/x, Axis trans: None 26, 24, 22, 2, 18, 16, Response 14, 12, 1, Quantify Compound Summary Report 8, 6, 4, Compound 2: Terfenadine Name Type Std. Conc RT Area µg /ml Dev UPLC_ZQ_12 Standard UPLC_ZQ_13 Standard UPLC_ZQ_14 Standard UPLC_ZQ_15 Standard UPLC_ZQ_16 Standard UPLC_ZQ_17 Standard UPLC_ZQ_18 Standard UPLC_ZQ_19 Standard UPLC_ZQ_2 Standard , Concentration (µg/ml) Figure 3: Excellent linearity is demonstrated for Terfenadine.

25 DECEMBER 25 ACQUITY UPLC SYSTEM 25 std UPLC_ZQ_ pg/µl each 1 pg on column 2-s peak widths Trimipramine : SIR of 2 Channels ES UPLC_ZQ_12 1: SIR of 2 Channels ES Terfenadine UPLC_ZQ_12 1: SIR of 2 Channels ES TIC Time Figure 4: UPLC MS limits of detection demonstrated with 1 pg on column for the analysis of Trimipramine and Terfenadine. Injection: 2 L Mobile Phase A:.1 formic acid in H 2 O Mobile Phase B:.1 formic acid in acetonitrile Gradient: 5 to 1 B in.5 min MS Conditions MS System: ZQ 2 Mass Detector Scan Range: 15 7 Da Scan Time:.11 s Dwell Time: 5 ms Interscan Delay:.2 s. Cycle Time:.13 s Ionization Mode: ESI Method 2 (See Figures 3 5) LC Conditions LC System: ACQUITY UPLC System Column: ACQUITY UPLC BEH C 18, 2.1 mm 5 mm, 1.7 m Flow Rate:.85 ml/min., 4 L split flow to MS Injection: 2 L Mobile Phase A:.1 formic acid in H 2 O Mobile Phase B:.1 formic acid in acetonitrile Gradient: 1 B to 9 B in 3 min MS Conditions MS System: ZQ 2 Mass Detector Scan Range: 15 7 Da Scan Time:.11 s Dwell Time: 5 ms Interchannel Delay:.2 s Interscan Delay:.2 s Cycle Time:.14 s Ionization Mode: ESI SIR Mode: m/z 295 (for Trimipramine) and m/z 472 (for Terfenadine) Results MS Spectral Quality Narrow Peaks Separations were accomplished in less then 3 s and MS spectral quality allows easy identification of all compounds (Figure 2). This is an indication of what can be achieved with UPLC and a single quadrupole MS in a high throughput laboratory when faster chromatography is the objective. Excellent Linearity Any analytical method requires a linear response relative to analyte concentration. In Figure 3, excellent linearity of response for Terfenadine is demonstrated for the UPLC MS method. Lower Limits of Detection The detectability of low amounts (pg) is also required. The combination of the enhanced resolution and peak concentration afforded by ACQUITY UPLC separations and the sensitivity and selectivity of the ZQ 2 mass detection enables the reliable detection of minor constituents (Figure 4).

26 26 ACQUITY UPLC SYSTEM DECEMBER 25 Quantify Compound Summary Report Compound 1: trimpiramine # Name Sample Text Type Std. Conc RT Area ug/ml Dev UPLC_ZQ_21 std 7 Standard UPLC_ZQ_22 std 7 Standard UPLC_ZQ_23 std 7 Standard UPLC_ZQ_24 std 7 Standard UPLC_ZQ_25 std 7 Standard UPLC_ZQ_26 std 7 Standard Ave: Std dev: RSD: Compound 2: terfenadine # Name Sample Text Type Std. Conc RT Area ug/ml Dev UPLC_ZQ_21 std 7 Standard UPLC_ZQ_22 std 7 Standard UPLC_ZQ_23 std 7 Standard UPLC_ZQ_24 std 7 Standard UPLC_ZQ_25 std 7 Standard UPLC_ZQ_26 std 7 Standard Ave: Std dev: RSD: Figure 5: UPLC MS reproducibility for Trimipramine and Terfenadine. Precision of Quantitation The quality of UPLC separations coupled with the high scan rate capabilities of the ZQ yield exceptional analysis precision (Figure 5). Conclusion The robust UPLC MS platform yields exceptional throughput, speed, and sensitivity for routine analyses. As the expectation to produce more quality data in less time becomes standard, the unique combined capabilities of the ACQUITY UPLC System with the ZQ 2 Mass Detector provide more resolution faster, thus allowing overall throughput to be increased without compromising data quality. This superior data is achieved without the need to resort to very high analytical flow rates, which compromises sensitivity. As a result of this optimized performance, this system solution delivers the uptime, throughput and productivity necessary for any high throughput analytical laboratory.

27 DECEMBER 25 ACQUITY UPLC SYSTEM 27 Enabling Significant Improvements for Peptide Mapping with UPLC Jeffrey R. Mazzeo, Thomas E. Wheat, Beth L. Gillece-Castro, and Ziling Lu Waters Corporation, Milford, Massachusetts Peptide mapping continues to be the preferred technique for the comprehensive characterization of biopharmaceutical products. Its applications include: the identification of proteins based on the elution pattern of peptide fragments, the determination of post-translational modifications, the confirmation of genetic stability, and the analysis of protein sequences when interfaced to a mass spectrometer. In a peptide map, it is necessary to resolve each peptide fragment into a single peak. Therefore, peptide mapping represents a significant chromatographic challenge, due to the inherent complexity of protein digests. In addition to the large number of peptide fragments that are generated from the enzymatic digestion of a protein, the number of alternative peptide structures (e.g., post-translational modifications, oxidations, etc.) can be significant. The capabilities of Ultra Performance LC (UPLC ) technologies make higher resolution peptide mapping possible. This application note demonstrates the advantages of UPLC for peptide mapping. Experimental LC Conditions LC System: Waters ACQUITY UPLC System Columns: UPLC: ACQUITY UPLC Bridged Ethyl-siloxane/silica Hybrid (BEH) C 18 ; and mm, 1.7 m HPLC: BioSuite PA-A mm, 3 m; and BioSuite PA-B mm, 3.5 m Flow Rate:.1 or.3 ml/min Mobile Phase: A:.2 TFA or.1 FA in H 2 O B:.18 TFA or.1 FA in ACN Gradient: Linear gradients from 5 5 B, times as indicated Temperature: 4 C Injection Volume: 2 or 5 L Detection: ESI MS Samples: MassPREP Peptide Standard Mixture; MassPREP Enolase Digest; Tryptic digest of -1 acid glycoprotein MS Conditions MS System: Waters Micromass Q-Tof micro Mass Spectrometer Software: MassLynx 4. Software, SP µl/min 2.1 mm column 25 µl/min 2.1 mm column.2 Increasing Resolution H (cm).15.1 Optimum resolution 3.5 µm packing 1.7 µm packing u (cm/s) Increasing Flow Rate Figure 1: van Deemter plot of a 15 Da peptide.

28 28 ACQUITY UPLC SYSTEM DECEMBER µl/min Peak 1 3 µl/min 33 µl 1 µl/min 2 µl 2 45 µl 33 µl 3 39 µl 25 µl TIC µl/min µl 27 µl 27 µl 3 µl 36 µl 27 µl 19 µl 18 µl 18 µl 19 µl 2 µl 18 µl TIC µl 24 µl Time (min) Figure 2: Effect of flow rate on the MassPREP peptide standard mixture. Both chromatograms are plotted to the same intensity scale. Results and Discussion The chromatographic benefits of UPLC are largely derived from reduced band broadening that is, in turn, a consequence of reduced diffusion distances in small particles. This process is quantitatively described in the van Deemter equation that relates height equivalent of a theoretical plate to linear velocity. This relationship is shown graphically in Figure 1 for a peptide of 15 MW on 3.5 m and 1.7 m packings. The minimum in the curve corresponds to the maximum efficiency, and greatest resolving power, for each particle size. At linear velocities, or flow rates, above and below the optimum, resolving power declines. As expected, the smaller particles have higher resolving power at a higher linear velocity. In quantitative terms, the 3.5 m particles have a minimum plate height of 8.11 m at a linear velocity of.17 mm/s. In contrast, a minimum plate height of 3.94 m is observed at.33 mm/s with the 1.7 m particles. In practical terms, these principles suggest that the small particles used in UPLC could double the resolving power in a peptide mapping experiment and could simultaneously reduce the separation time because the optimum is achieved at a higher linear velocity. For the 3.5 m particle, the optimum linear velocity corresponds to a flow rate of about 24 L/min. on a 2.1 mm i.d. column. In practice, such a flow rate would never be used for a peptide map because the separation times would be far too long. It is common practice to operate at a higher flow rate, typically about 25 L/min. on 2.1 mm columns. This linear velocity of about 1.7 mm/s corresponds to a plate height of about 21 m. This loss of resolution with a 1-fold increase in separation speed has come to be an accepted compromise. For 1.7 m particles, resolution is much better preserved at the higher flow rate. These chromatographic principles suggest several ways to approach improving peptide maps using UPLC. First, the smaller particle packing will improve both resolution and sensitivity by reducing diffusion-related band broadening. Second, the reduced plate height is consistent with obtaining the same or better resolution with shorter columns and higher flow rates. Third, the compromise between separation time and resolution will be more favorable with the smaller particles. The influence of volumetric flow rate on peptide separation performance with 2.1 mm i.d. columns was investigated. A standard peptide mixture was separated on a UPLC column run at 1 L/min and at 3 L/min, as shown in Figure 2. Flow rate, or linear velocity, was the only variable because the gradient change/column volume was the same, ensuring that

29 DECEMBER 25 ACQUITY UPLC SYSTEM ± ± ± ±.24 TIC 3.54± Time (min) Figure 3: UPLC retention time reproducibility for peptide mapping Traditional C µm 2.1 x 1 mm TIC ACQUITY UPLC BEH C µm 2.1 x 1 mm TIC Time (min) Figure 4: Suitability of UPLC for peptide mapping.

30 3 ACQUITY UPLC SYSTEM DECEMBER 25 1 BEH C µm 2.1 x 5 mm TIC Traditional C µm 2.1 x 1 mm, 3 Å TIC Time (min) 4. Figure 5: Reduced column length for increased speed. the chromatographic selectivity is constant. In experimental terms, a 75-min gradient was used at 1 L/min and a 25-min gradient at 3 L/min. To compare the results, peak volumes, calculated by multiplying the flow rate by peak width at the base, are shown in TIC the inset. Running at 1 L/min. provides, on average, about a 1/3 reduction in peak volume. 1 As expected from the chromatographic principles described above, peptide peak volumes, and, therefore, resolution and sensitivity, are optimum at 1 L/min. The flow rate is low compared to what is traditionally used for TIC peptide mapping with 2.1 mm i.d. HPLC columns. The commonly accepted flow rate represents a compromise between resolution and run time 1 but it also reflects instrumental limits in reproducibly pumping liquid at flow rates less than 15 L/min. with accurate and precise gradients. The ACQUITY UPLC instrument performs extremely well at a flow rate of 1 L/min. in gradient mode. This performance is demonstrated by the overlay of 6 gradient runs of a peptide standard, shown in Figure 3. The average and standard deviations of retention times are listed for each peak Deamidated T Time (min) Figure 6: Separation of a deamidated peptide from its native form. T-16 T-16 The chromatographic principles also suggest ways to reduce the run time of a peptide map. Peptide maps run by HPLC often require cycle times as long as 3 5 h to separate all the peptides within the digest, especially for large proteins like antibodies. While faster peptide maps would be desirable, it is critically important that resolution not

31 DECEMBER 25 ACQUITY UPLC SYSTEM FA Time TFA Figure 7: Effect of mobile phase modifier in UPLC BEH material. be compromised. The test results must provide the same level of information. The van Deemter equation predicts that plate height will be 2 4 fold less with 1.7 m particles than with 3.5 m particles. The same resolving power can, therefore, be obtained with a shorter column. These experiments focus on the physical aspects of the separation as they relate to band broadening. Successful peptide mapping depends, of course, on the interaction among the peptides, the mobile phase, and the column surface chemistry. In Figure 4, the separation of a tryptic digest of enolase is shown with a 3.5 m C 18 HPLC column with 3Å pores, typical of the most common peptide separation columns, and a 1.7 m UPLC column. Conditions are the same for both columns. In the UPLC separation, more peaks are observed. The overall resolution and sensitivity are higher. In the UPLC map, there are several small peaks that are difficult to discern with the HPLC run. This result demonstrates that UPLC offers higher resolution and sensitivity when compared to HPLC under the same gradient conditions. As is always observed when comparing two different column chemistries, the separations are not identical in every detail. The overall appearance of the chromatograms is, however, similar. This suggests that the selectivity of the UPLC column is suitable for peptide mapping. To show how UPLC can resolve the same number of peaks in a peptide map as HPLC but in less time, the separation of an enolase digest was done on a 5 mm length UPLC column with a 2-min gradient and on a 1 mm length HPLC column with a 4-min gradient, both with flow rates of 1 L/min. These chromatograms are shown in Figure 5. The UPLC separation shows the same number of peaks and a similar overall elution pattern as the HPLC separation, but in half the time. UPLC offers the potential to reduce cycle times for peptide maps. The higher resolution and sensitivity with UPLC is particularly important when the peptide map is used to detect modified peptides. Higher resolution ensures that modified peptides are resolved from the unmodified form, as well as from other peptides in the digest. Higher sensitivity means that modified peptides can be detected at lower levels. For example, in Figure 6, UPLC is used to separate a deamidated peptide from its unmodified form. UPLC should be the technique of choice for detecting all the peptides in a sample. Peptide mapping is frequently interfaced with electrospray ionization mass spectrometry (ESI MS) to provide additional information about the eluting peptides, including molecular weight and sequence. MS can also identify modified peptides and glycosylation sites. Therefore, it is important that a peptide mapping technique work well under conditions that are favorable for ESI MS. TFA is commonly used as an acidic additive for peptide maps with UV detection, but it can lead to suppression of ionization and reduced sensitivity in ESI MS. Formic acid is more desirable for LC MS, as it causes less ion suppression than TFA. However, many reversed phase columns used for peptide mapping show lower retention and broader peaks with formic acid than with TFA. In Figure 7, the separation of several peptides with formic acid is compared with TFA on a UPLC column with MS detection. With formic acid, the peak heights are

32 32 ACQUITY UPLC SYSTEM DECEMBER 25 1 SIC Time Figure 8: A glycopeptide separation. about 3-fold higher. There is only a slight reduction in retention, corresponding to a difference of a few percent organic at the point of elution, and a slight increase in peak width. This result indicates that the UPLC columns perform extremely well under conditions that are best for ESI MS. Glycosylation is an important post-translational modification that plays a critical role in determining the efficacy and safety of a therapeutic protein. Glycosylation can be analyzed on the intact protein by mass spectrometry, as released glycans or as glycopeptides in LC MS peptide maps. When glycosylation is characterized with LC MS of the glycopeptides, the site of attachment can be directly determined and structural information can be obtained through MS MS experiments. This approach is limited, however, by the poor chromatographic peak shape of glycopeptides and incomplete resolution of glycoforms with HPLC peptide mapping. The poor peak shape has been attributed to the large size of the glycopeptides and their heterogeneous structure. Figure 8 shows the UPLC MS separation of a tryptic digest of -1 acid glycoprotein. The MS detection was performed with a Q-Tof mass spectrometer, which is well suited for glycopeptides due to its extended mass range. Data is plotted as a selected ion chromatogram for m/z 657, a signature ion for glycopeptides resulting from carbohydrate fragments. The glycopeptides are detected as sharp, symmetrical peaks with UPLC. These characteristics are important for minimizing spectral overlap of different glycoforms of the same peptide. UPLC with ESI TOF mass spectrometry will be a powerful tool for studying the glycosylation state of proteins. that of common reversed phase HPLC peptide mapping columns and can be easily transferred to alternative modifiers that give better sensitivity in ESI MS. UPLC with ESI TOF is especially suitable for the separation of glycopeptides. The ACQUITY UPLC System is clearly proving to be the next-generation tool for peptide mapping. Conclusions UPLC facilitates notable improvements in peptide mapping when compared to HPLC. Better resolution is obtained in combination with generally increased sensitivity. Run time can be reduced without compromising resolution by reducing column length and by increasing flow rate. Selectivity is comparable to

33 DECEMBER 25 ACQUITY UPLC SYSTEM 33 Analysis of Prostaglandins with the Quattro Premier XE Kate Yu and Donald Kwet Waters Corporation, Milford, Massachusetts The accurate and precise measurement of candidate pharmaceuticals in biological fluids, environmental pollutants in soils and water or the measurement of toxins in various food matrices requires highly sensitive and specific assays. Over the past 1 years, LC MS and LC MS MS using multiple reaction monitoring (MRM) has become the technique of choice for the trace analysis of analytes in complex mixtures. This has been made possible by the advent of atmospheric pressure ionization sources, such as ESI and APCI. The highly sensitive and specific nature of the tandem quadrupole mass spectrometer facilitates simple method development and highly selective assays. The Waters Micromass Quattro Premier XE mass spectrometer with its T-Wave (1) technology has been specifically designed with high sensitivity analysis in mind. This new mass spectrometer incorporates enhanced electronics and a new high sensitivity detector for improved performance. The superior negative ion sensitivity of the Quattro Premier XE is illustrated using the analysis of three prostaglandins (D 2, E 2, and F 2a ), which are acidic steroidal compounds. Increasing Negative Ion Sensitivity The negative ion sensitivity of the Quattro Premier XE was compared to that of the Quattro Premier. The prostaglandin compounds were analyzed by LC MS MS using the Waters ACQUITY UPLC System. A 5 L aliquot of the sample was loaded onto an ACQUITY UPLC BEH mm C 18, 1.7 m column. The column was eluted with a gradient of 1 95 B over 2 min at.8 ml/min, where A 1 acetonitrile in.1 formic acid and B 8/2 acetonitrile/methanol. The column eluent was monitored by negative ion ESI, using the MRM transitions for prostaglandins D 2 and E 2, and the MRM transition for prostaglandin F 2a. The resulting XIC chromatograms for prostaglandin F 2a using the Quattro Premier XE and Quattro Premier are displayed in Figure Here we can see that with the Quattro Premier XE peak area of the analyte is increased by a factor of approximately 1 compared to the Quattro Premier. A 9-fold increase in peak area was obtained for prostaglandins E 2 and 1-fold for D 2 (data not shown). Improving the Limit of Detection To further illustrate the superior performance of the Quattro Premier XE, the Limit of Detection (LOD) was compared to that of the Quattro Premier for both Prostaglandin D 2 and Prostaglandin E 2. The resulting data is displayed in Figure 2. It is shown that with the same on-column loading, the peak-topeak signal-to-noise value obtained from the Quattro Premier increased from 9.4 to 47.1 for Prostaglandin D 2 (left) and from PGF2a, 353.5>193.1 Peak area = 6 PtP S/N = MRM of 1 Channel ES e Peak area = 59 PtP S/N = MRM of 1 Channel ES e Time Peak area increased 9.8 times Peak-to-Peak Signal-to-Noise (PtP S/N) increased 5.4 times Figure 1: Comparative response for prostaglandin F 2a on the Quattro Premier (top) and Quattro Premier XE (bottom).

34 34 ACQUITY UPLC SYSTEM DECEMBER 25 PGD2, > MRM of 1 Channel ES- PGE2, > MRM of 1 Channel ES- 2.63e3 3.2e3 PtP S/N = 9.36 QP PtP S/N = MRM of 1 Channel ES MRM of 1 Channel ES- 2.63e3 3.2e3 PtP S/N = PtP S/N = QP-XE Time Peak area increased 9.7 times Time Peak area increased 8.8 times Figure 2: Comparing the LOD of the Quattro Premier (top) and Quattro Premier XE (bottom) to 35.7 for Prostaglandin E 2 (right). Therefore, from this data we can ascertain that the new Quattro Premier XE produces a 3- to 5-fold increase in signal-to-noise and a 1-fold increase in peak area sensitivity. Conclusion The new Quattro Premier XE mass spectrometer provides the ultimate in sensitivity in both positive and negative ion modes. This performance is further enhanced by the T-Wave-enabled fast scanning and fast MRM switching capabilities. The Quattro Premier XE, when combined with the superior chromatographic performance of the ACQUITY UPLC makes this the ideal LC MS MS platform for high sensitivity, high throughput analysis. Notes (1) The traveling wave device described here is similar to that described by Kirchner in US Patent 5,26,56 (1993).

35

36 721417EN