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1 column Waters A Reprint from Spring 99 If you would like a subscription, or more information, please contact your nearest Waters Office. Check the Waters website for up-to-date information: Published by Waters Corporation Maple Street Milford, MA 0 USA Tel: Toll-free: Fax: Editor: Uwe D. Neue, Ph.D. ISSN # Waters Corporation

2 Role of Anionic Ion-Pairing Reagents in the Development of Pharmaceutical Methods: Water-Soluble Vitamins Dorothy Phillips and Walter Foster Introduction Symmetry reversed-phase columns have been shown to exhibit excellent peak-shape for acidic, basic and neutral compounds without the need for mobile phase additives to suppress silanol activity (). In this paper, we show that these advantages also hold, when anionic ion-pairing reagents are used to control the retention of basic analytes. Pharmaceutical methods are often developed for the separation of a mixture of acidic, basic and neutral compounds. The most frequently used method for these separations is reversed-phase chromatography. The parameters varied to achieve these difficult separations are ph, temperature, ionic strength, organic modifiers and ion-pairing reagents. The charge and relative hydrophobicities of the components of a mixture determine the range of the parameters that can be used for optimizing a separation. Mobile phase ph strongly affects the retention of ionic or ionizable components. The neutral components will shift in position mainly due to changes in the organic modifier. Both cationic and anionic ion-pairing reagents can be used to change selectivity by modifying the retention of charged compounds. At a working ph where these reagents are completely ionized, they interact with oppositely charged species in the sample. Thus, the interaction is an attraction between a positive and a negative charge. The ion-pairing reagents can interact with the solute either in the mobile phase or after adsorption on the stationary phase. It has been reported that ion-pairing reagents are adsorbophilic ions (having an affinity for the stationary phase), and no measurable evidence for ion-pair formation in the mobile phase was found (). Some investigators have treated paired-ion chromatography as a dynamic ion-exchange while others favor a mixed mode of dynamic ion exchange and adsorption, with one mechanism dominating over the other depending on the experimental conditions. (,, ) Ion-pairing agents have been shown to have negligible effect on neutral species but will affect all charged species in the sample matrix. Cationic reagents cause the largest shift in the response factor of acidic or opposite charged species with significantly less effect on similarly charged basic molecules. Anionic reagents behave similarly and have the strongest effect on basic compound with the opposite charge. It has been shown that at concentrations between 0 and mm/l, ion-pairing reagents have the greatest effect on the retention factor, k, of compounds with similar charge. This effect suggests that modes of interaction other than electrostatic occur between these reagents and the solute.() The nature of the pharmaceutical compounds in a mixture can be determined by their response to ion-pairing reagents. At low ph, the retention of strong bases is increased, whereas that of weak bases is less affected. At neutral ph, the retention factors of strong acids, but not weak acids, are changed by ion-pairing reagents. () The aim of this paper is to show how anionic reagents have their greatest effect on the retention factor of basic compounds. The most commonly used anionic reagents are sodium pentane-, hexane-, heptane-, and octanesulfonate and octylamine hydrochloride. Sodium hexane sulfonate was used in this report. Experimental A Waters Symmetry C 8 column (.9 mm x 0 mm) was used for the chromatography. The seven water-soluble vitamins included riboflavin, niacinamide, thiamine, pantothenic acid, pyridoxal, pyridoxamine and pyridoxine. The aqueous component of the mobile phase was 0.% phosphoric acid with varying concentrations of sodium hexane sulfonate; methanol was present at a constant % (v/v). The chromatography was done on a Waters HPLC system comprised of the Model 00 Solvent Delivery System, the Model 90 Multiwavelength Detector and the Model Autosampler. The Millennium Chromatography Manager was used for system control, data acquisition and analysis. Results and Discussion The resolution of seven water-soluble vitamins was optimized by varying the concentration of sodium hexane sulfonate (Waters PIC Reagent B) from mm to 0 mm. The retention times of those components that interact with the pairing reagent changed while those that did not remained constant. The structures of the vitamins are given in Figure. The chromatography was done at ph. where the acidic vitamins are not charged but the basic vitamins are. Figure shows the chromatography for the vitamins at four different concentrations of sodium hexane sulfonate. The first two peaks in

3 Figure are niacinamide and pantothenic acid which are unaffected by the presence of the hexane sulfonic acid. Figure, which plots retention time versus concentration of pairing reagent, shows the relatively flat lines for niacinamide and pantothenic acid. These two vitamins do not pair with hexane sulfonic acid. Figure : Structures of Water-Soluble Vitamins CONH. Niacinamide HOCH C(CH ) C CONH(CH ) COO H. Calcium Pantothenate Ca The three pyridoxine bases exhibit the retention factor shifts typical for ion-pair chromatography. The strongest changes in retention are observed at low concentration of the ion-pairing reagent. Above a concentration of about 0 mm/l, the increase in retention levels off. Therefore, a method that uses a concentration of the ion-pairing reagent above 0 mm/l is more rugged than a method using lower concentrations. Pyridoxine and pyridoxamine shifted more than pyridoxal with increasing molarity of sodium hexane sulfonate as shown in Figure. The pyridoxamine with a primary amine, paired with hexane sulfonic acid to a higher degree than the other pyridoxine bases, thus showing the greatest increase in retention time. The selectivity between pantothenic acid and pyridoxal increases because only the latter shifts in retention time with increased molarity of the pairing agent. The neutral vitamin riboflavin shows only a 0% change in retention time with a fourfold increase in ion-pairing reagent. The retention of riboflavin actually decreases instead of increasing as pointed out in Figure. The decrease in retention time of riboflavin could be due to the ion-pairing agent creating a more hydrophilic environment on the stationary phase, leading to less interaction of this vitamin with the surface. Figure : Chromatography of Water-Soluble Vitamins as a Function of the Concentration of Ion-Pairing Reagent N CH HOCH CHO. Pyridoxal CH N HCI HO CH CH Column: Symmetry C 8.9 mm x 0 mm Mobile Phase: Methanol/0.% phosphoric acid with PIC B : Flow Rate: 0.mL/min Detection: 0 nm Sample: 0 µl of mixture with component at µg/ml, at µg/ml, at µg/ml, at µg/ml, at µg/ml, at µg/ml, and at µg/ml mm/l PIC B, Peaks. Niacinamide. Pantothenic acid. Pyridoxal. Pyridoxine. Pyridoxamine. Niboflavin. Thiamine. Pyridoxine Hydrochloride N CH.HC HOCH CH NH 0 mm/l PIC B 8. Pyridoxamine Dihydrochloride CH CH H C N N O H C N NH O. Riboflavin CH N NH S CH CH N N + CH CH C - HC mm/l PIC B 8 8. Thiamine Hydrochloride 0 mm/l PIC B 8

4 Details: Details:. Detritylated failures and protecting groups Details: 0 0 = AMQ = NH = Val = Asp 8 = Arg = Met = Ser 9 = Thr = Lys = Glu 0 = Ala = IIe = Gly = Pro = Leu = His = Tyr 8 = Phe DMT blocked failures 8. AU units ( mgs) of oligonucleotide Moffat, A.S. 988 (Nov/Dec). Researchers Pursue Anti-Sense Technology In Quest for Novel Drugs and Agriproducts, Genetic Engineering News. Vol 8. Pages and. Klauser. A Antisense Start-Ups Surveyed, Bio/Technology. Vol 8. Pages 0-0. Thiamine which is a basic compound showed the largest shift in retention time, because it interacts strongly with hexane sulfonic acid. Thiamine becomes more retained, shifting its retention time from.8 to.9 min. As the concentration of ion-pairing reagent is increased, thiamine becomes more retained than its neighboring peak riboflavin. This change in elution order is a beautiful example of the control of selectivity that ion pairing reagents contribute to the tool-kit of the methods development chemist. Deputy et al did an extensive study to develop a rugged and reliable HPLC method for separation of water-soluble vitamins. They were able to develop a method to separate four vitamins (niacinamide, pyridoxine, folic acid, riboflavin and thiamine) using 0 mm sodium hexane sulfonate. () Figure : Retention Factor versus Concentration of Ion-Pairing Reagent Retention Factor, k Concentration of Sodium Hexanesulfonate (mm/l) Niacinamide Pantothenic Acid Pyridoxal Pyridoxine Pyridoxamine Figure : Retention Factor of Vitamins versus Ion-Pairing Reagent Concentration.0. Riboflavin Thiamine Retention Factor, k Concentration of Sodium Hexanesulfonate (mm/l) Rapid Polyolefin Additive Separation Using Nova-Pak C 8 Waters Applications Notes Analysis of Hydrolyzed Bovine Serum Albumin Using Waters AccQ Tag Method for Amino Acid Analysis Absorbance, 00nm HPLC Purification of Phosphorothioated DNA for Antisense Therapeutic Investigations Fluorescence Response mv DMT blocked full length product 0 0 Absorbance 0 nm Column: Bondapak HC8 HA (8 x 00mm Eluent A: 00mM ammonium acetate, ph./methanol (9:, v/v) Eluent B: Methanal/Milli-Q water (9:, v/v) Sample: Phosphorothioated mer (DMT Injection: mls, containing O.D.0nm units ( mgs) of oligonucleotide in 0% A and 0% B, injected into Column: Bondapak HC8 HA (8 x 00mm Eluent A: 00mM ammonium acetate, ph./methanol (9:, v/v) Eluent B: Methanal/Milli-Q water (9:, v/v) Sample: Phosphorothioated mer (DMT Injection: mls, containing O.D.0nm units ( mgs) of oligonucleotide in 0% A and 0% B, injected into HC8 HA reversed-phase material in an 8 x 00mm Radial-Pak Column: Bondapak HC8 HA (8 x 00mm Eluent A: 00mM ammonium acetate, ph./methanol (9:, v/v) Eluent B: Methanal/Milli-Q water (9:, v/v) Sample: Phosphorothioated mer (DMT Injection: mls, containing O.D.0nm HC8 HA reversed-phase material in an 8 x 00mm Radial-Pak cartridge. This same packing is available in a variety of column dimension for gram scale purifications as required for antisense therapeutic investigations in 0% A and 0% B, injected into Waters HC8 HA reversed-phase material in an 8 x 00mm Radial-Pak cartridge. This same packing is available in a variety of column dimension for gram scale purifications as required for antisense therapeutic investigations. Moffat, A.S. 988 (Nov/Dec). Researchers Pursue Anti-Sense Technology In Quest for Novel Drugs and Agriproducts, Genetic Engineering News. Vol 8. Pages and. Klauser. A Antisense Start-Ups Surveyed, Bio/Technology. Vol 8. Pages 0-0. APPLICATIONS NOTES For your free Applications Note File Folder, please check box on the reply card and return today.

5 PIC Reagents Ordering Information Workable UV Range in Mobile Quantity Description Phase (nm) (Vials/Pkg) Part No. Price PIC A (tetrabutylammonium hydrogen sulfate) Low UV PIC A 00+ WAT0889 $0.00 PIC A (tetrabutylammonium phosphate) 0+ WAT PIC B (pentane sulfonic acid) Low UV PIC B 00+ WAT PIC B 0+ WAT PIC B (hexane sulfonic acid) Low UV PIC B 00+ WAT Pic B 0+ WAT PIC B (heptane sulfonic acid) Low UV PIC B 00+ WAT PIC B 0+ WAT PIC B8 (octane sulfonic acid) Low UV PIC B8 00+ WAT PIC B8 0+ WAT PIC Sample Kit (One vial each: PIC A, B, B, B and B8) WAT Reagent D (dibutylammonium phosphate) + WAT08.00 Conclusion The vitamin assays shown here give an example of the power of ion-pairing reagents for the separation and analysis of pharmaceutical mixtures. The methods development chemist can use the ability of basic compounds to interact with the reagent to increase the retention time for these bases. The retention of neutral compounds remains essentially unaffected. We used this method to separate seven vitamins via a rapid isocratic method using a Symmetry C 8 column. References. U. D. Neue, D. J. Phillips, T. H. Walter, M. Capparella, B. Alden and R. P. Fisk, LC-GC, (99), 8. B. A. Bidlingmeyer, S. N. Deming, W. P. Price, Jr., B. Sachok and M. Petrusek, Retention Mechanism for Reversed- Phase Ion-Pair Liquid Chromatography, J. Chromatogr. 8 (99) 9-.. Lin and Cantrell, Anal. Chem. (99) 0-0. K. G. Wahlund, and A. Sokolowski, Reversed-Phase Ion-Pair Chromatography of Antidepressive and Neuroleptic Amines and Related Quaternary Ammonium Compounds, J. Chromatogr. (98) M. T. W. Hearn, in Ion-Pair Chromatography, Theory and Biological and Pharmaceutical Applications, Chromatographic Sciences Series, M. T. W. Hearn (Ed), Vol., Marcel Dekker, Inc., New York, 98. G. K. C. Low, A. Bartha, R. A. H. Billet, L. de Galan, Use of Gradient Scans in Reversed Phase Ion-Pairing Chromatography in Connection to Optimization Strategies, International Symposium on Column Liquid Chromatography, HPLC 8, Amsterdam.. A. L. Deputy and L. P. Murray, Development and Optimization of an HPLC Separation for the Water Soluble Vitamins in A Multivitamin-Supplement, International Symposium on Column Liquid Chromatography, HPLC 9, Minnesota, USA. For more information, please check box 8 on the Business Reply Card and return today.