The Application of SepaFlash Gamma HILIC ARG Cartridge for the Purification of Strong Polar Thiazide Compound

Background introduction of application technology research center thiazide is a kind of heterocyclic molecule containing nitrogen and sulfur elements, which belongs to diuretic drugs and is commonly used to treat hypertension and edema (such as caused by heart failure, liver failure or renal failure) Thiazide diuretics reduce the risk of death, stroke, heart disease and heart failure due to hypertension [1] Thiazides were first discovered and developed by Merck in the 1950s The first thiazide drug, chlorothiazide, was listed in 1958 and sold under the trade name of diiril [2] In most countries, thiazides are the cheapest antihypertensive drugs [3] The mechanism of long-term hypotensive effect of thiazides is not clear When administered in an acute manner, thiazides reduce blood pressure by causing diuresis, decreased plasma volume, and decreased cardiac output However, after long-term administration, thiazides can reduce blood pressure by reducing peripheral resistance (i.e vasodilation) [4,5] Although the toxicity of thiazides is low, overdose or long-term use of thiazides may cause dose-dependent adverse drug reactions, including ion disorder, hyperuricemia, hyperglycemia, etc In this application case, the molecular structure of the sample contains the parent structure of thiazide, which is not suitable for the separation and purification of normal silica gel column or reversed-phase C18 column The application engineer of Santai technology uses sepaflash ® HILIC Arg column to prepare the chromatographic system sepabean ® machine with fast liquid phase It was successfully separated, and the target product meeting the purity requirements was obtained, which provided a convenient and efficient solution for the separation and purification of such samples In the experimental part, the sample to be purified in this paper comes from a pharmaceutical company It is an alkaline compound containing thiazine parent structure, with large polarity However, its solubility in methanol, water and other common solvents is low, and the purity of the crude product is about 81% (HPLC analysis result) See Figure 1 for the schematic diagram of its molecular structure Figure 1 Schematic diagram of molecular structure formula of thiazine samples due to the large molecular polarity of the sample, the solubility is very low in common normal solvents, so the normal phase separation mode is excluded first Next, we consider the separation and purification of it by reversed-phase C18 column In order to improve the solubility of the sample in water, a small amount of trifluoroacetic acid (TFA) was added to the sample to make the sample salt and dissolve in water Take a proper amount of sample solution and put it on the flash separation column with a syringe The flash separation and purification experimental parameters of the sample are shown in Table 1 Table 1 Sepabean ® machine t chromatographic column 12 g sepabelash ® C 18 reversed phase separation column (spherical silica gel, 20-45 μ m, 100 μ m, order No.: sw-52 22-012-sp) 120 g sepabelash ® HILIC Arg separation column, Order No.: sw-5622-120-sp) detection wavelength: 220 nm, 54 nm mobile phase solvent A: water (add 0.1% TFA) solvent B: acetonitrile (add 0.1% TFA) flow rate 15 ml / min 50 ml / min injection volume 100 mg 500 mg elution gradient time (min) solvent B (%) time (min) solvent B (%) 0 100 70 The results of 10.09035.00 and the flash preparation map of the sample on the common C18 reversed-phase column are shown in Figure 2 It can be seen from Fig 2 that, due to the large polarity of the sample, the sample is basically not retained on the common C18 reversed-phase column As the mobile phase is directly eluted from the separation column, the effective purification cannot be obtained Fig 2 As we all know, the flash preparation map of the sample on the C18 reversed-phase column, the highly hydrophilic polar stationary phase is used in the hydrophilic interaction chromatography (HILIC), and the proportion of water phase in the mobile phase gradually increases during the elution process, which is suitable for the separation and purification of the samples with larger polarity Therefore, the hydrophilic mode was considered to separate and purify the sample Sepaflash ® HILIC Arg separation column introduced by Santai technology has a strong polar Arg group bonded on its packing surface (see Figure 3), which can produce enough retention effect on hydrophilic compound samples The flash preparation map of the sample on the HILIC Arg separation column is shown in Figure 4 It can be seen from Figure 4 that under the HILIC separation mode, the sample is well preserved on the stationary phase of the separation column, and other impurities in the crude product are also effectively separated The purity of the purified product is about 95.3% (see Figure 5) after the collected components are freeze-dried and analyzed by HPLC, which can be used for the next research and development Fig 3 Schematic diagram of bonding phase on the packing surface of sepaflash HILIC Arg separation column Fig 4 Flash preparation map of sample on HILIC Arg separation column Fig 5 HPLC analysis map of target product Therefore, for the preparation and purification of thiazine strong polar compounds, sepaflash ® HILIC Arg separation column with sepabean ® machine The rapid liquid preparation chromatographic system provides a fast and efficient solution for the rapid separation and purification of such samples Sepaflash? HILIC Arg separation column series products launched by Santai technology have a variety of specifications (see Table 2) Table 2 Sepaflash ® HILIC Arg separation column parameters (packing: high efficiency spherical Arg, 20 – 45 μ m, 100 Å ） Item Number Column Size Sample Size Max.Pressure (psi/bar) SW-5 6 22-004-SP 5.4 g 5.4 mg –  108 mg 400/27.5 SW-5 6 22-012-SP 20 g 2 0  mg –  0.40 g 400/27.5 SW-5 6 22-025-SP 33 g 33g –  0.66 g 400/27.5 SW-5 6 22-040-SP 48 g 48 mg –  0.96 g 400/27.5 SW-5 6 22-080-SP 105 g 105 mg –  2.1 g 350/24.0 SW-5 6 22-120-SP 155 g 155 mg –  3.1 g 300/20.7 SW-5 6 22-220-SP 300 g 3 0 0 mg –  6.0 g 300/20.7 SW-5 6 22-330-SP 420 g 42 0 Mg – 8.4 g 250 / 17.2 for more detailed specifications of sepabean ® machine or ordering information of flash purification column, please visit CBG online store: https://store.chembiango.com/ Reference 1 Wright JM, musini VM (July 2009) Wright, James M, ed "first line drugs for hypertention" Cochrane Database syst Rev 8 (3): cd001841 2 Beyer KH (1993) "Chlorothiazide How the thiazides evolved as antihypertensive therapy" (PDF) Hypertention 22 (3): 388 – 91 3 Whitworth JA, World Health Organization, international Society of Hypertension Writing Group (November 2003) "2003 World Health Organization (WHO)/International Society of Hypertension (ISH) statement on management of hypertension" (PDF) J Hypertens 21 (11): 1983–92 4. Hughes AD (2004) "How do thiazide and thiazide-like diuretics lower blood pressure?" J Renin Angiotensin Aldosterone Syst 5 (4): 155–60 5. Zhu Z, Zhu S, Liu D, Cao T, Wang L, Tepel M (2005) "Thiazide-like diuretics attenuate agonist-induced vasoconstriction by calcium desensitisation linked to Rho kinase" Hypertension 45 (2): 233–9.