Tài liệu HPLC for Pharmaceutical Scientists 2007 (Part 8C) pdf

the flow rate (mL/min) to determine the time (in minutes) needed. Therefore, the lower the flow rate, the longer the equilibration time. Some typical equilibration times for various column dimensions are shown in Table 8-7; however, these should only be used as a guide. If complete equi- libration is not achieved, early eluting components may show differences in retention from run to run. An experiment could be run such that three dif- ferent methods could be run with different equilibration times. For example, if a 15-cm × 4.6-mm i.d. column and a flow rate of 1mL/min was used, then the equilibration times for the three methods would be 5 min (3 CV), 9min (5 CV), and 11 min (6 CV) equilibration times, respectively. If the retention of the early eluting components are consistent (less than 1% variation in reten- tion time) in all three methods, then the lowest equilibration time could be used. However, if the early eluting components show greater variation in their retention time with the 5-min equilibration time compared to the methods with the 9- and 11-min equilibration time, then an equilibration time of greater than 5 min is warranted. Optimization of the optimal equilibration time is required for reproducible methods. Other considerations include differences in dwell volumes from the differ- ent HPLC systems.The dwell volume should be determined for all the systems in the laboratory and based on these determinations, this should be factored into the calculation of the equilibration time. For example, if the maximum dwell volume of all the systems in a particular laboratory to which the method is transferred to is 2 mL and you are running on an instrument at 1 mL/min that has a dwell volume of 1 mL, then you should add an extra minute of equi- libration time. This becomes extremely important during method transfers where the instruments in the receiving laboratory may be different. 8.5 METHOD DEVELOPMENT APPROACHES 8.5.1 If Analyte Structure Is Known Determine if analytes are acidic, basic, or neutral. This will allow the chromatographer to choose a pH such that the analyte is being analyzed METHOD DEVELOPMENT APPROACHES 385 TABLE 8-7. Equilibration Times for Columns of Different Dimensions Length (cm) Diameter (cm) Column Volume (CV) (mL) 3 CV (mL) 5 CV (mL) 15 0.46 1.74 5.2 8.7 15 0.3 0.74 2.2 3.7 10 0.46 1.16 3.5 5.8 10 0.46 1.16 3.5 5.8 10 0.21 0.24 0.7 1.2 5 0.46 0.58 1.7 2.9 5 0.3 0.25 0.7 1.2 predominately in one ionization state. Use the rules for pH shift and pK a shift to ensure that the analyte is one predominant ionization state and choose the appropriate mobile-phase pH (see Chapter 4). Some general guidelines are as follows: If your target analyte is an acidic analyte (pK a ≥ 3), use a 0.2 v/v% phosphoric acid mobile phase. If target analyte is a basic analyte (pK a ≥ 7–9), use an ammonium acetate buffer (pH 5.8) to analyze in its ionized form or use a 25 mM ammonium hydroxide buffer (pH 10.5) or 25 mM N-methyl pyrrolidine buffer (pH 10.5) to analyze in its neutral form. Use a 10-cm × 3.0-mm column packed with 3-µm particles and intermediate polarity phase such as a C8 column that is stable for the pH at which you may be running the probe separation. Run in the gradient mode using an acidic buffer or a basic buffer from 5% to 95% of organic component, or up to the buffer solubility limit over 10 min, and use an isocratic hold for 10 min to ensure elution of hydrophobic components. Use a flow rate of 0.8–1 mL/min flow rate and 40°C temperature. Injection volume should be on the order of 5–20 µL and the concentration of the analyte should be 0.5–1 mg/mL. This corresponds to approximately 5–20 µg injected on column. On the other hand, for neutral analytes higher analyte loading such as 50–100 µg maybe used since nonideal interactions with the stationary phase are less prevalent. Note that for ionizable compounds, especially basic compounds when analyzed in their ionized state, higher mass on column may lead to mass overload of “hot spots” on the bonded phase and poor peak efficiencies may be observed. Try not to load more than 10 µg on column for basic compounds. Usually greater loading capacity is obtained for basic compounds when they are analyzed in their neutral state. Note that for columns with larger inner diameters such as 4.6 mm, larger sample loads may be acceptable. Once the probe gradient is run, check the diode array purity; and if LC-MS is available, run as well to check for peak homogeneity. If you have any known precursors or impurities, run them as well to ensure resolution from the main component and to make sure they are adequately retained. The main analyte should elute between k 2–5. If the main component elutes at k = 2–5 and is spectrally pure and the impurities all elute k > 1, the method is complete. If the retention factor of the impurities is below 1, then an isocratic hold at the initial organic composition should be implemented until the minor component (impurity) elutes k > 1 and then a linear gradient can be implemented. The method could be further optimized by increasing the flow rate as long as the backpressure limitation of the system has not been reached. A general rule of thumb is that the backpressure should not exceed 85% of the maximum back- pressure for a particular HPLC system. If resolution is not achieved between a critical pair, the use of a shallower gradient can be investigated. If that does not increase the resolution, then a longer column (15-cm column, packed with 3-µm particles of the same sta- tionary phase type) should be used with a reduced flow rate of 0.7 mL/min (due to backpressure limitations). 386 METHOD DEVELOPMENT If separation is still not achieved, consider using a different organic modi- fier such as mixture of MeCN and methanol to possibly induce changes in the selectivity. Also, the wavelength of detection must be considered, especially if MeOH is used due to its UV cutoff (absorbs <210 nm). If methanol does give you desired selectivity, then an analyst needs to determine if sufficient response (S/N > 10 :1) is obtained at desired LOQ (i.e., 0.05% solution of target), especially if the wavelength for detection is <210nm. If changing the organic modifier does not work, consider changing the mobile-phase pH (analyze the molecule in a different ionization state). For example, if a basic compound was originally analyzed under basic conditions (pH >> pK a ), try to use acidic conditions (pH << pK a ) with the acetonitrile in the initial gradient. If that still does not work, then consider using a different stationary phase (phenyl or polar embedded) employing the initial gradient, with initial aqueous mobile phase and acetonitrile organic modifier, and repeat the process that was performed on the original column used for initial method development. The final method optimization may include varying the gradi- ent slope, column temperature, and flow rate. Note that multiple pH values and columns can be screened in gradient mode at the same time as well. This will increase the efficiency/probability of obtaining the best column/conditions and the best demonstrated chromato- graphic selectivity. Note that the aqueous phase pH values that would be chosen for these pH/column screening studies should be based on knowledge of the physicochemical properties of the molecule, taking into consideration the mobile-phase pH and analyte pK a shifts in the hydro-organic media. 8.5.2 If Method Is Being Developed for Separation of Active and Unknown Component Define the criteria for the method such as the LOQ, maximum run time, wave- length detection, and so on. Look at the structure of the target analyte (esti- mate pK a ) or use ACD (advanced chemistry development) and determine the best pH to run the method. Try to use shorter columns for gradient scouting experiments (5 cm × 4.6mm ) packed with 3-µm columns or use a high- pressure system (max pressure 15,000 psi) with 10-cm × 2.1-mm, 1.7-µm parti- cles. Use 35–45°C as starting temperature. If pH scouting studies are needed, run a probe linear gradient using 0.2 v/v% phosphoric acid on a short column (5-cm × 4.6-cm column) to determine the isocratic conditions for the pH studies. Run pH studies isocratically to determine the desired pH region to understand the behavior of the impurities in the analyte mixture. The desired pH region of the aqueous phase is the pH region where the retention of the components in the mixture do not significantly change their retention as a function of the pH of the aqueous phase. Track impurities using diode array if possible. Run a linear gradient at a pH within the desired pH region and hold at high organic concentration on 5-cm × 4.6-mm column. If you obtain sufficient resolution, then you are finished. If you need more METHOD DEVELOPMENT APPROACHES 387 resolution, then use a 15-cm × 3-mm i.d. column. If resolution is obtained, then you are finished. If desired resolution/selectivity is not obtained, then screen different organic modifiers/different stationary phase types. Note that the separation of the critical pair may be obtained on an alternate stationary phase that offers additional selectivity. In addition to the weak dispersive types of interaction that are available on a C8 or C18 phase, phenyl phases may provide additional interactions such as π–π-type interac- tions and may assist in providing additional selectivity. If the impurities/active are very polar, the use of polar embedded phases may provide additional selec- tivity by introduction of a secondary type of interaction such as hydrogen bonding close to the surface in the organic-enriched layer. Alternatively, for basic compounds, different counteranions could be introduced in the mobile phase in order to increase retention of protonated basic amines. These are known as chaotropic reagents and were discussed in Section 4.10. This may lead to increased retention and increased selectivity between critical pairs of components. Moreover, if the laboratory has an automated method development system, then this could be used to determine the best set of gradient conditions to give the best resolution between the critical pair or pairs on multiple columns. When using an automated method development system such as AMDS (Waters, MA) for gradient optimization, generally two types of organic mod- ifiers are used at two different temperatures employing a steep/shallow gra- dient on two to six columns. Based on these scouting runs and the users’ acceptance criteria for the method, a resolution map is generated by input of the data into Drylab, Chromsword, ACD, or another program. From this res- olution map the best conditions are chosen and optimized (change in flow rate, multistep gradient ramps, etc.), and these conditions are run to confirm that the method that was predicted is indeed representative of the actual separa- tion. This is typically called a verification run. AMDS relies on constraints of the DryLab model. Note that Drylab is not suitable for the following types of compounds: • Chiral compounds • Achiral isomers or diastereomers • Inorganic ions • Carbohydrates • Proteins and peptides The DryLab model utilized in Waters AMDS has additional requirements:The number of sample components should not exceed 12; peak area% should be greater than 1%. These requirements are necessary to achieve greater predic- tion accuracy only. Any discrepancies could be corrected manually in DryLab using the data entry screen by manually entering the retention of the compo- nents from the scouting runs (to assign the peaks with a certain number). DryLab has been used for the method development of model drug candidates 388 METHOD DEVELOPMENT and their degradation products, by optimization of temperature and gradient slope, and the historical review on the milestones and concepts in the devel- opment of DryLab software is given in references 20–23. 8.5.3 Defining System Suitability System suitability parameters with their respective acceptance criteria should be a requirement for any method. This will provide an added level of confidence that the correct mobile phase, temperature, flow rate, and column were used and will ensure the system performance (pump and detector). This usually includes (at a minimum) a requirement for injection precision, sensitivity, standard accuracy (if for an assay method), and retention time of the target analyte. Sometimes, a resolution requirement is added for a critical pair, along with criteria for efficiency and tailing factor (especially if a known impurity elutes on the tail of the target analyte). This is added to ensure that the column performance is adequate to achieve the desired separation. System suitability requirements for retention time, efficiency, resolution, and tailing factor are set based on prior method challenging experiments and prior method development experience. This is a dynamic process; and as the user gains more experience with the method, the breadth of the acceptance criteria is further expanded until the method is finally validated for the intended purpose. Two examples are given for setting system suitability requirements for challenging separations. In the first example, if a separation is to be carried out where the retention of the target analyte may have a greater propensity to vary with slight changes in pH, tighter controls for the pH requirement should be implemented, where the pH of the aqueous phase should be controlled to ±0.05 units. Moreover, some preliminary experiments should be performed using an aqueous mobile-phase pH ±0.2 units from the desired pH to determine if this will have an effect on the critical pairs in the separa- tion and what the desirable retention time window is. This information is useful to define the system suitability criteria for the method. Also, it is rec- ommended to run the separation on different lots of columns to see if there is any lot-to-lot variability. Preferably, running the separation on columns that were made from different batches of base silicas is desirable. Also, obtaining columns from different synthetic bonding batches made on the same batch of silica is also desirable. In the example shown in Figure 8-22 for a drug product that contains two actives, three different columns from three different lots of base silica were used and the pH of the aqueous mobile phase was varied from 5.7 to 6.1, with the target pH being 5.9. Some of the specific system suitability parameters and acceptance criteria that were set included tailing factors (5% peak height), retention time windows for peaks A and B, and sensitivity requirement. Some of the selected system suitability parameters were set to the following: METHOD DEVELOPMENT APPROACHES 389 System Suitability Parameters • Tailing factor (5% peak height) for peak B ≤ 1.5 • Tailing factor (5% peak height) for peak A ≤ 1.5 • Rt for peak A must be 12.0 ± 1.3 min • Rt for peak B must be 21 ± 1.0 min • The S/N of the LOQ solution (0.05%) for both actives A and B must be ≥10:1 In the second example, if it is known that a potential degradation product can occur and will elute close to the active, a resolution requirement should be set for this critical pair.When trying to set a resolution requirement between crit- ical pairs of impurities, standard samples containing the critical pair should be readily available. However, standard samples may not be available with all critical impurities so the standard may be spiked with authentic impurities. If authentic impurities are not available or are in limited quantity, then the drug substance may be degraded in solution using mild stress conditions to produce a decomposition product or products that can be used to define a resolution requirement for a critical pair. The mild stress conditions should produce decomposition products in situ in a fast time scale. In the following example in Figure 8-23, the drug substance was stressed with 3% hydrogen peroxide for 1 hr at 25°C and 80°C to generate impurity A. At 80°C, suitable degrada- tion was obtained to determine the resolution requirement between impurity A and the active B (target analyte). This requirement was set because it was postulated that this drug substance could be readily oxidized. Indeed in solid state stability studies, minor amounts of the impurity A (oxidized impu- rity) were observed under accelerated conditions (40°C/75% RH, 3 months). 390 METHOD DEVELOPMENT Figure 8-22. Waters XBridge 150- × 3.0-mm, 3.5-µm C18 column. Column temperature 40°C [(A) 90%: 20 mM ammonium phosphate buffer: 10% MeCN, (B) 100% MeCN]. Gradient: 10% A to 85% B over 38 min. Flow: 0.6 mL/min. Another example in regard to in situ degradation for generation of a system suitability sample is given in the literature [24]. 8.5.4 Case Study 1: Method Development for a Zwitterionic Compound Method development for the analysis of a zwitterionic drug substance by reversed-phase HPLC was undertaken.The zwitterionic compound A contains an acidic functionality, ( w w pK a 4.0) and a basic functionality ( w w pK a 3.0). Both of these pK a values were determined using ACD Labs (Advanced Chemistry Development, Toronto, Canada) software. Given this information, the chro- matographer could apply the pH and pK a selection rules (including pH and pK a shifts) outlined in Sections 4.5 and 4.6 in Chapter 4 to select the optimal pH to work at in order to avoid working near the pK a values of either of the ionizable functionalities. The following case study will illustrate (a) why working at pH values at or near the pK a values of the API will lead to sepa- rations that may not be robust and (b) what influence the pH has on the inher- ent retention of intermediate compound A and related synthetic by-products. These experiments could be conducted as an exercise to further understand the effect of pH on the retention of the species in the sample of interest since the synthetic by-products may have different ionizable functionalities then the parent compound (intermediate). METHOD DEVELOPMENT APPROACHES 391 Figure 8-23. In situ degradation for generation of system suitability solution. 8.5.4.1 Gradient Screening. An initial method development was performed using a Phenomenex Luna C18 (2) column with acetonitrile as the organic mobile-phase component, and the aqueous portion was a 10 mM ammonium monohydrogen phosphate buffer adjusted to pH 2 with phosphoric acid. Ini- tially, a linear gradient was used from 60% to 80% MeCN with a hold at 80% MeCN for 10 minutes. An early eluting component was observed close to the void volume using this probe gradient. Also, no peaks were seen to elute during the 80% MeCN isocratic hold. Therefore, a new gradient method (shown in Figure 8-24) with an initial isocratic hold to retain the more polar species and removal of the latter isocratic hold at 80% MeCN was used. The new method employed an isocratic hold at 50% MeCN for 5 min, and then a linear gradient was run from 50% MeCN to 80% MeCN from 5 to 25 minutes. Note that a 150- × 4.6-mm column was used, but a 150- × 3.0-mm could have been easily used with proper adjustment of the flow rate. 8.5.4.2 pH Screening Study. Once the probe gradient method is selected, a pH study can be conducted. The pH study in gradient mode was carried out using 10 mM ammonium monohydrogenphosphate as a buffer. The w w pH of the aqueous portion of the eluent was adjusted to 2, 3, 4, 5, 6, and 7 with phos- phoric acid. Phosphate is not a buffer at pH 4 and 5, but this is only used for the pH screening experiment. In the event that pH 4 or 5 was deemed accept- able for the separation, a suitable buffer that has buffering capacity in that 392 METHOD DEVELOPMENT Figure 8-24. w w pH study on zwitterionic compound A on a Phenomenex Luna C18 (2) column. Method conditions are indicated in the figure. region would be chosen. Note that upon changing the w w pH of the mobile phase, at least 25 column volumes of the new mobile phase were passed through the column prior to sample analysis. This step should be a general requirement when performing w w pH scouting studies. An alternative approach is to perform repeat injections of the intermediate after changing the mobile phase w w pH until consistent retention times are obtained for all components in the mixture, which would deem that the column is adequately equilibrated. As can be seen from Figure 8-24, the retention of all of the impurities and the API are dependent on the pH of the mobile phase. The intermediate and the related impurities exhibited lower retention at low pH. Retention increased initially with increasing pH, where it reached a maximum and then decreased as the pH was further increased. The optimal pH range to carry out further method development was determined to be w w pH 6–7, where the reten- tion of the API and related impurities did not change as a function of the w w pH. The intermediate and its related impurities are zwitterionic in nature and contain both acidic and basic functionalities; this is confirmed by its bell- shaped dependence on the mobile-phase pH (Section 4.5 in Chapter 4). Peak X elutes before the main component at low w w pH (2.0), but it elutes after the main component at pH values higher than 2.0 ( w w pH 3–7). 8.5.4.3 Peak Tracking. During method development, if it is observed that critical pairs are changing elution order, the use of diode array and/or LC-MS should be employed to assist in peak tracking. In this particular example com- paring the elution of impurity X and the intermediate, it was believed that the elution order had switched at w w pH 2 and w w pH > 3.1. Therefore the reversal of elution order was determined by comparing the diode array spectrum of impu- rity X and was further confirmed by LC-MS (note that ammonium bicarbon- ate mobile phase was used for pH 7 LC-MS analysis and TFA was used for pH 2 LC-MS analysis, with both using ESI in the positive ion mode). Note that the diode array profiles of impurity X did not directly overlay at pH 2 and pH 7, and an isobestic point (where two substances absorb at a certain wave- length of light to the same extent) was observed that can be attributed to changes in conjugation of the aromatic ring when analyzed at different pH values (see Section 8-6 for more information on the effect of pH on changes in UV absorbance). Peak tracking at different pH values by diode array some- times is a challenging task, especially if the analyst wants to compare the UV spectrum of the impurity present at different ionization states. This was the driver to perform LC-MS analysis in order to confirm the [M + H] + ion of this impurity species. Indeed, when LC-MS analysis was performed, it was con- firmed that this impurity had shifted elution order when the pH of the mobile phase was changed from 2 to 7. An extracted ion spectrum of the [M + H] + ion of impurity X at pH 7 was performed for facile identification of the impurity. 8.5.4.4 Anomalies During Method Development. Further evaluation of the chromatograms in Figure 8-24 revealed that some late eluting peaks were METHOD DEVELOPMENT APPROACHES 393 observed with the pH 5, 6, 7 mobile phases, and those peaks were not observed when the lower pH mobile phases were used. In order to troubleshoot if these peaks are indeed present in the sample or artifacts, it should be determined if the late eluting peaks are (1) synthetic process impurities with different ion- izable functionalities or (2) impurities formed in the sample solvent (indicat- ing lack of solution stability). In order to make this assessment, the stability of the intermediate in the diluent was challenged. In this case study the solu- tion was stored at room temperature under normal light conditions and the diluent was acetonitrile. The experiments for w w pH 2–4 were performed on day 1, and those for w w pH 5–7 were performed on day 2 (≈36 hr after initial prepa- ration).A further investigation was performed by preparing a fresh stock solu- tion and storing one-half of the solution in the refrigerator (4°C) for 36 hr while the other half of the solution was stored in a clear volumetric flask on the bench (ambient conditions), and it was determined that these impurities are actually formed in the diluent at room temperature under normal light conditions (see Section 14.8.1 for further details). The solutions were deter- mined to be light-sensitive. The case study message is that fresh solutions should be prepared daily in amber volumetric flasks and a tray cooler should be used when possible when the stability of the sample in solution has not yet been determined. 8.5.4.5 Method Selectivity and Choice of Column. Generally during method development, multiple columns at various pH values can be screened in isocratic or gradient using a column switcher or commercially available method development systems that have the ability of running five or more columns. The reason is that different stationary-phase types may provide a dif- ferent selectivity and give the chromatographer additional confidence in resolving potential co-eluting species. In this case study, the separation per- formed on a Luna C18(2) (Phenomenex, Torrance, CA) was compared to the separation performed on a polar end-capped column, Synergy-Hydro-RP (Phenomenex, Torrance, CA). Similar trends in the retention dependence rel- ative to w w pH were observed for all impurities and intermediate on both types of columns, since the effect of pH on the analyte retention is a function of the analyte ionization state (Figures 8-24 and Figure 8-25). However, differences in selectivity and differences in the magnitude of the retention can be related to stationary-phase type and surface area of the column, respectively. Differ- ences in selectivity were observed between peak X and impurity Y at w w pH 2 when comparing these two columns. The Hydro-RP column showed greater selectivity at w w pH 2 between impurity Y and impurity X. Note that the late eluting degradation products present in Figure 8-24 at w w pH 5–7 were not observed using this column, since the samples were stored protected from light. It was determined that the storage conditions were important to mini- mize the degradation product formation. Although similar retention profiles were obtained at w w pH 6 and 7 (desired pH range for the separation) on both columns, the pH stability of the Phenomenex Luna C18 (2) (up to w w pH 10) is 394 METHOD DEVELOPMENT [...]... path for initial mobilephase pH selection In the following case study for this pharmaceutical compound M, the method development scenario and rationale for each iteration in the method development process is highlighted Also, a method development flow chart for gradient separations is included which can be used as a general strategy for method development (Figure 8-37) References are made in this case... also available to allow for in-silico prediction of the analyte pKa Also, using selected fragments of the molecule can also be helpful for pKa determination of the desired molecule because the pKa values for each of these fragments of the molecule may be readily available from the literature This is only an estimate at best, but can guide the chromatographer down the right path for initial mobilephase... temperature Method development for ionizable analytes requires a judicious choice of the mobile-phase conditions and system parameters in order to perform the analysis of the compounds in their desired ionization state Choosing the optimal parameters in the “chromatographer’s toolbox” allows for the development of rugged and reproducible methods 8.5.6 Case Study 3: Method Development for a Diprotic Basic Compound... para and ortho isomers at three different pH values (w pH 2, w w w wpH 8, and wpH 8.6) were determined The optimal pH for the separation is at w pH 2 However, studies were performed at w pH 8, and w pH 8.6 to illustrate w w w why working at these higher pH values would not be ideal for the separation from a robustness point of view Although favorable changes in selectivity may occur at a pH near the... in their more neutral form with increasing amount of organic component in the mobile phase, making them more hydrophobic However, the increase in the organic leads to a decrease in the analyte retention for both isomers due to a decrease in the analyte hydrophobicity Two effects that are acting upon the retention of the isomers in opposite directions could provide an explanation for the curvature in... because the methanol adsorbs in a form of a monomolecular layer Hence, due to the difference in the analyte partition coefficients and their adsorption on the stationary phases, changes in selectivities could be obtained for two components when using either methanol/water or acetonitrile/water eluents Varying the type of organic modifier in RPLC separations is recommended for the separation of isomers 404... when performing method development and choosing the column to perform further method optimization experiments, especially when working at higher pH values and/or higher temperatures 8.5.4.6 Method Optimization Having established that a monohydrogen diammonium phosphate buffer with w pH adjusted to 7.0 (with phosphoric w acid) was the best mobile-phase pH to use (no variation in retention of peaks for w... in MeCN that was stored for 4 days at room temperature under normal light conditions (Figure 8-26) As the temperature was increased from 15°C to 40°C, the resolution between an impurity eluting on the tail of the intermediate and the intermediate increased, as did the resolution for the potential degradation products eluting later in the separation Also, the tailing factor for the intermediate decreased... 80 v/v% acetonitrile The hold at higher organic concentration is usually employed for compounds during early development in the event that more hydrophobic species are formed either during the processing (i.e., change in synthetic routes, hold point stability, etc.) or during solid-state stability studies of the active pharmaceutical ingredient or drug product 8.5.5 Case Study 2: Influence of pH, Temperature,... isomers in opposite directions could provide an explanation for the curvature in the plot of these ionizable species Also, this could account for the change in selectivity with an increase in % organic component at w pH 8.6 (Figure 8-30) This should not be a pH for further method develw opment, since the method would not be robust in regard to slight changes of w w wpH However, it was shown that although . factor (5% peak height) for peak B ≤ 1.5 • Tailing factor (5% peak height) for peak A ≤ 1.5 • Rt for peak A must be 12.0 ± 1.3 min • Rt for peak B must be 21. diluent was acetonitrile. The experiments for w w pH 2–4 were performed on day 1, and those for w w pH 5–7 were performed on day 2 (≈36 hr after initial prepa-