ing the method development proce

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During the method development process, separation of coeluted peaks can be fine-tuned with infinite combinations of mobile phase factors, including solvent type, pH, additives, and operating conditions (such as temperature, flow rate, and gradient time). LCGC North Am.31(8), 612621 (2013). A. Loo, Int. Column: 150 mm x 2.1 mm HALO Peptide ES-C18; flow rate: 0.3 mL/min; temperature: 60 C, gradient: 247% acetonitrile in 40 min; detection: MS,(+)TIC 3001800 m/z, 4 kV, with a Shimadzu MS-2020 SQ. Basic analytes are ionized at low pH, though most basic drugs have sufficient hydrophobicity for adequate reversed-phase LC retention. A universal HPLC method using a sub-3-m charged surface hybrid (CSH) column in the analysis of 12 new chemical entities (NCEs). Orlando, J. Biomol. There is also a common practice to use the same levels of additives in both mobile phases A and B, such as 0.1% trifluoroacetic acid in acetonitrile when using 0.1% trifluoroacetic acid (TFA) in water as mobile phase A. Technically, there are no perceivable differences between 100% acetonitrile or 0.1% trifluoroacetic acid in acetonitrile for separation reproducibility or elution order in reversed-phase LC except for reducing gradient shifts with UV detection at low wavelengths (to be covered in a later section). A common practice is to date all mobile phases and use aqueous buffers for one week, simple acidified water (such as 0.1% formic acid or trifluoroacetic acid) for 12 months, and organic solvents for at least 3 months. Reversed-phase liquid chromatography is the dominant mode in high performance liquid chromatography (HPLC) for quantitative analysis, and is used in ~80% of all HPLC applications (1-3). Boyes, W. Miles and B. Libert, J. Biomol. This practice can be problematic because trace contaminants adhering to the electrodes (such as preservatives from the pH calibration buffers to allow room temperature storage) can cause substantial ghost peaks in gradient elution (see the example in Figure 5). Another class of additives used to increase retention and selectivity of basic analytes is inorganic chaotropic agents (such as PF6-, BF4-, ClO4- ions) that form neutral ion pairs under acidic conditions in reversed-phase LC (1,2). Tetrahydrofuran is rarely used in reversed-phase LC, despite its strong solubilizing power and eluotropic strength. A,1217(6), 858880 (2010). Second, it would overwhelm all MS signals in the positive ionization mode. Figure 6 illustrates the comparative performance of four mobile phase additives (formic acid, ammonium formate/formic acid, trifluoroacetic acid, and difluoroacetic acid) in the HPLCUVMS analysis of a mixture of five peptides. (7) D. V. McCalley, J. Chromatogr. (26) B.E. Figure 2: A case study in column screening using acidic and basic mobile phases in the gradient separation of a test mixture consisting of 12 acidic (red), basic (blue) and neutral (black) drugs. The stationary phase provides retention, and, in conjunction with the mobile phase, differential migration of the solutes. Volatile buffers are labeled with an asterisk are used for LCMS applications. Figure 8 shows an example of a protein separation using trifluoroacetic acid compared to difluoroacetic acid and formic acid in mobile phase A. Method transfer can be less problematic with simplified methods using easily prepared binary mobile phases. The traditional mobile phase A for protein and peptide separations is 0.1% trifluoroacetic acid, which has ion-pairing capability for better peak shapes and offers reasonable UV sensitivity at 210220 nm for peptide bonds. (18) D.W. Hill and A.J. Hewitson, P.C. Methods 10(10), 989992 (2013). Chetwyn, LCGC North Am. Recent research by Boyes and associates has uncovered two promising additives that deliver a good compromise of performance for peak shape and MS sensitivity: difluoroacetic acid and 3-fluoropropionic acid (26). Toxicity and safety issues due to peroxide formation are problems preventing its widespread use except in gel permeation chromatography. In such cases, periodic checks of the pH and blank injections can verify the integrity and cleanliness of the mobile phase preparations. 33(6), 402-413 (2015). Comparisons of mobile phase buffers at elevated pH conditions revealed significant effects on silica solubility and expectations for stability under high pH and temperature conditions (16). An acidic pH of 24 is used for most pharmaceutical applications. Comparisons of peptide separations with inline MS detection shows trifluoroacetic acid strongly suppressed ionization, difluoroacetic acid much less so, with formic acid or ammonium formate exhibiting highest signal, whereas trifluoroacetic acid, difluoroacetic acid, and ammonium formate show excellent peak shape. Anecdotal evidence suggests that the advantages of difluoroacetic acid as a replacement are more apparent for ESI signal intensities of peptides and smaller proteins compared to larger proteins up to the size of immunoglobins. Fischer, G. Goldberg, and P.C. Column: 150 mm x 2.1 mm HALO Protein 400 C4; flow rate: 0.3 mL/min, temperature: 80 C; gradient: 2838% acetonitrile in 30 min. A,779(1), 29-71 (1997). Long-chain alkyl sulfonates (C5 to C12) combine with basic solutes under acidic pH conditions to form neutral "ion-pairs" that are retained in reversed-phase LC. Dimethyl sulfoxide has been suggested as an additive for improving MS detection in high complexity or high sensitivity proteomic sample LCMS analyses (27,28). 34(6), 408419 (2016). (27) F.E. Looking at the historical practice of HPLC, there appears to be a trend toward the use of simpler mobile phases for several practical reasons. Paradoxically, innovation in reversed-phase LC centers on new instruments and columns while scant research efforts are placed on the development of newer reagents for mobile phases. All rights reserved. An added benefit of this approach is to yield columns usable at high pH (such as the Agilent Poroshell HPH-C18) (20). The example shown is for a mixture of 5 synthetic peptides, resolved in 10 mM of each acid or acid plus salt (in the case of ammonium formate). Pure water can be used for the separation of neutral molecules. Most practitioners prefer acetonitrile because of its strong eluotropic strength, low viscosity (0.37 cP) leading to higher column efficiency, and good UV transparency (to 190 nm). The use of 0.1% phosphoric acid appears to be under-utilized because there is a traditional preference for phosphate buffer. The Use of Balanced Absorbance Mobile Phases. Chaotropes are better suited for gradient analysis and can produce lesser baseline shifts, but are not MS-compatible. Here, we discuss those and other trends and best practices, as well as the fundamentals behind them. The role of the mobile phase in controlling retention and selectivity in reversed-phase LC for neutral and ionizable solutes has been discussed extensively in textbooks, and the reader is referred to them for a detailed discussion (13). These simple mobile phases at 0.1% v/v are prepared by pipetting 1.0 mL of the acid into 1 L of purified water, and can be used directly without further filtration. He was an adjunct professor of chemistry at the University of Georgia (Athens, GA), and continues a long-term collaboration with Professor Ron Orlando at the Complex Carbohydrate Research Center in Athens. Iraneta, and D. Morrison, Waters Corporation, Milford, Massachusetts, 720003720EN, Sep. 2010. 33(10), 764775 (2015). Reduced Usage of Silanol-Masking, Ion-Pairing and Chaotropic Reagents. Figure 4: Chromatograms illustrating gradient shift problems encountered during method development of a stability-indicating method for a drug substance using (a) 0.1% trifluoroacetic acid in mobile-phases A and B and (b) balanced absorbance mobile phases of mobile-phase A of 0.05% trifluoroacetic acid in water and mobile-phase B of 0.03% trifluoroacetic acid in acetonitrile. Second, the use of simpler binary solvents with linear gradient segments increases method robustness through reduction of method transfer issues for regulated assays (9). Dong, LCGC North Am. At that time, many bonded phases had such acidic silanols that basic solutes would not elute without the use of silanol-masking reagent additives in the mobile phase (such as triethylamine). The CSH C18+ column shows excellent peak shapes for all NCEs, while the other column displays considerable peak tailing for four of the more basic NCEs. The effect of these additives on MS system stability and maintenance are still unknown. 2, C. Horvth, Ed. (11) K. Schug and T. Taylor, The Essential CHROMacademy Guide: Mobile Phase Optimization Strategies for Reversed Phase HPLC, Webcast, http://www.chromacademy.com/essential_guide_webcast/mobile_phase_optimization_strategies_for_reversed_phase_hplc/mobile_phase_optimization_strategies_for_reversed_phase_hplc.pdf (accessed on 6/27/2018). Dong and R.M. Figure 1 shows a chromatogram of a test mixture containing two active pharmaceutical ingredients (APIs) spiked with process impurities and degradants for an MS-compatible stability-indicating assay of a combinational drug product (3). In this installment, we present an overview of modern trends and best practices in mobile-phase selection and preparation in reversed-phase LC, with a focus on pharmaceutical applications and increasing peak shape and detection performance by UV and MS. (a) blank gradient; (b) sample run. Buffers are required to tightly control the pH of mobile phase A for critical assays. Buffers of ammonium salts of volatile acids are used for the development of MS-compatible HPLC methods. (14) D. Johnson, B.E. Technology (2018), in press. (c) The UV spectrum of 0.05% trifluoroacetic acid vs. water. Modern trends in gradient methods include the use of simple binary mobile phases (such as 0.1% formic acid or 0.1% ammonia) with high-efficiency columns packed with superficially porous particles and low-silanophilic bonded phases (8). Reversed-phase LC uses a hydrophobic stationary phase and a polar mobile phase, and analytes are retained primarily by hydrophobic interaction. (25) B. Boyes, S. Schuster, J.J. Kirkland, B. Wagner, B. Libert, J. DeStefano. The C18 column yielded excellent symmetrical peaks for all NCEs when used with a buffered mobile-phase A (such as 20 mM ammonium formate at pH 3.7). Triethylamine was frequently used in drug analysis to yield better peak shapes and acceptable batch-to-batch reproducibility in regulated testing (18). Overall Trends Towards Simpler Mobile Phases. However, it does add system dwell volume and is rarely used in today's laboratories. Boyes, and R.C. The retention in reversed-phase LC is highly correlated to solutes' log P or partitioning coefficients between water and octanol. Kuhlman, A. Apffel, S.M. A buffer is prepared by mixing a weak acid with the salt of its conjugate base (or a weak base with the salt of its conjugate acid). Mass Spectrom. Dong, LCGC North Am. Methanol has substantial UV end-absorbance below 210 nm. Newer additives (difluoroacetic acid and 3-fluoropropionic acid) as alternatives to trifluoroacetic acid for an increased MS sensitivity are suggested. Before the 1990s, silica-based columns could not be used with high pH mobile phases, because of the dissolution of silica support at pH values >8. Figure 5: A case study illustrating the potential issue of a ghost peak caused by dipping the pH electrode into mobile-phase A during pH adjustment. The development of hybrid particles and improved bonding chemistries for silica particles has extended the usable pH range of these specific columns to and range of 112 (210 at column temperatures >40 C) (3,15-16). Adapted with permission from reference 3. Toxicol.19, 233242 (1994). Column: 150 mm x 3.0 mm, 3.5-m Waters XTerra MS C18; mobile-phase A: 20 mM ammonium bicarbonate, pH 9.1; mobile-phase B: acetonitrile; gradient: 350% B in 40 min, 5080% B in 3 min; flow rate: 1 mL/min; temperature: 50 C. First, triethylamine may permanently alter the selectivity of the column. The internal filters in most HPLC pumps (replaced during preventive maintenance programs) appear to be adequate to allow successful routine HPLC operation without filtering in most laboratories. For this mAb, mobile phases using formic acid exhibit poor retention and peak shape, compared to either trifluoroacetic acid or difluoroacetic acid mobile phases. (16) J.J. Kirkland, M.A.van Straten, and H.A.Claessens, J. Chromatogr. The elimination of the filtration process and many less relevant mobile phase additives is also favored. Figure 7: Comparison of the separation of a tryptic digest of enolase (50 picomoles) on the same column, using different acid modifiers each at 10 mM concentration. Acetonitrile is an aprotic solvent and is a proton acceptor with capability for interaction (3). Note the excellent peak shapes of all peaks under high-pH conditions without any peak splitting. Such materials can retain highly polar bases and acids, with less reliance on hydrophobic counterion additives, ensuring better compatibility with MS detection, and faster re-equilibration between separations. Table II lists the common mobile phase additives with their respective pKa and UV cutoffs. To prevent such contamination, the analyst should dispense a small amount of mobile phase A into a vial (such as a scintillation vial) to check the pH and continue this iteration until the correct pH is reached.

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