pKa determination of oxysophocarpine by reversed - phase high performance liquid chromatography
© Huo et al.; licensee Springer. 2013
Received: 8 January 2013
Accepted: 24 May 2013
Published: 20 June 2013
In this study, a RP - HPLC method was applied for determination of pKa value by using the dependence of the capacity factor (k) on the pH of the mobile phase for oxysophocarpine (OSP).
The effect of the mobile phase composition on the ionization constant was studied by measuring the pKa value at different MeOH concentrations, ranging from 10 to 20% (v/v). Based on all pH - k curves plotted and pH values at inflection point calculated, experimental pKa value obtained for oxysophocarpine was 6.5.
This method was successfully applied to realize low sample consumption, rapid sample throughput, high sensitivity and precision.
KeywordsOxysophocarpine pKa value HPLC
Oxysophocarpine (OSP) is one of the active alkaloid ingredients in the traditional Chinese medical herb kuh-seng (Sophora flavescens Ait.). Kuh-seng has a long history of curing cancer, viral hepatitis, cardiac arrhythmia, skin diseases and so on, and the curative effects have been proved definitely clear. Alkaloids, such as matrine, oxymatrine, sophocarpine and oxysophocarpine (OSP) (Lin et al. 2010), are commonly known to present in kuh-seng as main active constituents. Previous study found OSP could effectively adjust the immunity and antiviral system, but the reports about its physicochemical property was just a little, especially the pKa value of OSP had not been determined.
To determine an ionization constant (pKa), is of great importance in many scientific areas, particularly in pharmacy. For instance, the lipophilicity, solubility and permeability of drug compounds are pKa dependent, so it is important to obtain the reliable information about pKa as early as possible during the drug development process (Wiczling et al. 2004). Factors talked above can define the fate of a drug in vivo, and the early-obtained information about drug absorption, distribution and transport processes may decrease costly the development process of a compound with poor pharmaceutical properties.
The commonly used techniques for pKa determination are potentiometry, conductometry and spectrometry (Narasimham & Barhate 2011; Foulona et al. 2007; Gonen & Rytwo 2009). The first two methods are accurate, but both exist high sample consumption problem. Comparatively, UV spectrometry is being of relatively higher sensitivity which requires a big difference of UV spectrograms between a compound’s acidic (HA) and its basic (A) forms being observed. In our pre-experiment, spectrograms of OSP at different pH buffer solution were obtained, but the wavelength shifts as a function of pH was not obvious. As a result, the suitable detection wavelength could not be chosen.
Recently, some scholars successfully applied HPLC method for determination of the pKa value for a compound (Padró et al. 2012; Demiralay et al. 2009; Wiczling et al. 2008; Wiczling et al. 2006; Pehourcq et al. 2006; Subirats et al. 2007; Miklautz et al. 2006). With analysis of the change in retention time (t R ) of analyte versus the pH environment change of the mobile phase, Horvάth et al. (1997) proposed the theoretical basis for studying the pH dependence of chromatographic retention for ionizable solute in liquid chromatography. According to this theory, Angelov T et al. estimated the pKa value of parabens by HPLC with a formula (1) and its conversion formula (2) (where k is the retention factor at a given pH of the compound investigated. k HA and k A are the retention factor of unionized and fully ionized forms, respectively), and the results showed the determined pKa value was close to the literature values (Angelov et al. 2008). Wei Zhang et al. applied above method to determine the pKa value of scutellarin. According to the retention factor of detective compound and corresponding pH values of mobile phase, they derived a curve equation of pH - k (Wei et al. 2010). Thanks to the curve fitting software, the pKa value was determined as the inflection point in the curve.
Consequently, in the light of therapeutical value of OSP and the importance of the pKa value for a compound, this paper firstly developed the HPLC method to obtain the pKa value of OSP, and further investigated the effect of different percentage of organic phase in MeOH - phosphate buffer mixtures, 10, 15, 20% (v/v), on the pKa value.
Chemicals and reagents
The standard substance of oxysophocarpine (purity about 98%) was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). HPLC grade methanol (Caledon Lab Ltd., Georgetown, Germany) was used for chromatographic separation. Phosphoric acid, sodium dihydrogen phosphate and disodium hydrogen phosphate were of analytical grade from Guangzhou Chemicals Co. Ltd. Deionized water used throughout the experiments was purified by Milli-Q system (Millilpore, Bedford, MA, USA).
The HPLC analysis was carried out on a Shimadzu LC-20A System with N2010 chromatographic software, LC-20AT pump, SPO-M20A diode array detector, SIL-20A Auto sampler and CT0-10AS column oven. An Ultimate®XB-C18 column (4.6 × 250 mm ID, 5 μm ) was used for the determination.
Measurement of the pH value of the mobile phase was done with a PHB-3 pH electrode.
Preparation of the phosphate buffers at different pH values
The different pH ranges of the phosphate buffers, between 2.0 to 9.5, were obtained by adjustment with sodium dihydrogen phosphate (0.01 mol/L), disodium hydrogen phosphate (0.01 mol/L) and phosphoric acid (0.01 mol/L).
The data of 20% ( v/v ) MeOH with calculation method
pH of mobile phase
Tailing factor of peak
pH ( i+ 1)–pH i
k ( i+ 1) – k i
(k ( i+ 1) – k i ) / (pH ( i+ 1)–pH i )
The data of 15% ( v/v ) MeOH with calculation method
pH of mobile phase
Tailing factor of peak
pH ( i+ 1)–pH i
k ( i+ 1) – k i
(k ( i+ 1) – k i ) / (pH ( i+ 1)–pH i )
Precision evaluation of chromatographic system
Five successive sample injections to LC column eluted by different mobile phase, such as MeOH - phosphate buffer (pH6.0, which was close to the inflection point in pH - k plot) mixture at 20:80 (v/v), 15:85 (v/v) and 10:90 (v/v), respectively, were operated to verify the precision of the chromatographic system. Then the relative standard deviation (RSD) of retention times (t R ) and peak areas (A) for OSP under different mobile phase conditions were calculated.
Results and discussion
Chromatographic condition and chromatogram behavior
The data of precision evaluation of chromatographic system (n=5, mean)
In the pH - k plot of a compound investigated, the pH value at the inflection point in the curve, which is the equilibrium point for acidic (HA) and basic (A) forms of the compound, corresponds to the pKa value of analyte (Angelov et al. 2008). Based on the formula (2) (Angelov et al. 2008) mentioned in above ‘Introduction’, this study ascertained the values (k, pH, k HA , k A ) by finding the point with maximal slope in the sigmoidal curve.
The pH - k plot of OSP was a sigmoid curve, as the pH value increased, the change trend of capacity factor (k) was steady - sharp rise - steady, as shown in Figure 2. Through calculating the value of (k i+1 – k i ) / (pH i+1 – pH i ), the slope of specific detected points in the curve had been calculated, as shown in Tables 1 and 2. In the formula (2), the pH and k value were defined as the corresponding point with maximum-slope. The k HA was the minimum value of the retention factor (k) , whose slope of corresponding point approximated zero, and similarly k A was the maximum one.
In the Table 1, when using MeOH - phosphate buffer at 20:80 (v/v) as mobile phase, the k HA , k A , pH and k required by the formula were determined as 0.844, 7.045, 7.6 and 6.626 respectively, so the pKa value of OSP was calculated as 6.46. In the Table 2, when using MeOH - phosphate buffer at 15:85 (v/v) as mobile phase, the k HA , k A , pH and k required by the formula were determined as 1.804, 9.932, 7.0 and 7.964 respectively, and the pKa value of OSP was calculated as 6.50. From these result, the organic modifier percentage in the mobile phase had no influence on the pKa detection. However, when the percentage of MeOH was lower than 10% (v/v), and the pH of mobile phase was above 7.5, OSP could not be eluted so as to hard to obtain a complete pH - k curve.
In this study, the RP - HPLC method was successfully applied to determine the pKa value of a compound investigated with low sample consumption, rapid sample analysis, high sensitivity and precision. Simultaneously, the ionization constant of OSP obtained from this test will play an important role in the field of pharmacokinetics and clinical treatment.
- Ando HY, Heimbach T: pKa determination by using a HPLC equipped with DAD as a flow injection apparatus. J Pharm Biomed Anal 1997, 16: 31-37. 10.1016/S0731-7085(97)00084-8View Article
- Angelov T, Vlasenko A, Tashkov W: HPLC determination of pKa of Parabens and Investigation on their lipophilicity parameters. J Liq Chromatogr Relat Technol 2008, 31: 188-197.View Article
- Demiralay EC, Alsancak G, Ozkan SA: Determination of pKa values of nonsteroidal anti-inflammatory drug-oxicams by RP-HPLC and their analysis in pharmaceutical dosage forms. Liquid Chromatography 2009, 32: 2928-2936.
- Foulona C, Duhalb N, Lacroix-Callensb B, et al.: Determination of pKa values of benzoxa-, benzothia- and benzoselena-zolinone derivatives by capillary electrophoresis Comparison with potentiometric titration and spectrometric data. Eur J Pharm Sci 2007, 31: 165-171. 10.1016/j.ejps.2007.03.002View Article
- Gonen Y, Rytwo G: Using a matlab implemented algorithm for UV-vis spectral resolution for pKa determination and multicomponent analysis. Analytical Chemistry Insights 2009, 4: 21-27.
- Lin Z, Huang CF, Liu XS, et al.: In vitro anti-tumour activities of quinolizidine alkaloids derived from Sophora Flavescens Ait. Basic Clin Pharmacol Toxicol 2010, 108: 304-309.View Article
- Miklautz H, Keller D, Lopez Holguin F, Woloszczak R: Automated determination of the pKa values of 4-hydroxybenzoic acid in cosolvent–water mixtures and related solvent effects using a modified HPLC system. Anal Bioanal Chem 2006, 384: 1191-1195. 10.1007/s00216-005-0278-0View Article
- Narasimham L, Barhate VD: Physico-chemical characterization of some beta blockers and anti-diabetic drugs-potentiometric and spectrophotometric pKa determination in different co-solvents. Eur J Chem 2011, 2(1):36-46. 10.5155/eurjchem.2.1.36-46.371View Article
- Padró JM, Acquaviva A, Tascon M, et al.: Effect of temperature and solvent composition on acid dissociation equilibria, I: Sequenced pKa determination of compounds commonly used as buffers in high performance liquid chromatography coupled to mass spectroscopy detection. Anal Chim Acta 2012, 725: 87-94.View Article
- Pehourcq F, Thomas J, Jarry C: HPLC retention date and the pKa values in a series of 2-Amino-2-Oxazolines. J Liq Chromatogr Relat Technol 2006, 20: 1381-1389.View Article
- Subirats X, Roses M, Bosch E: On the effect of organic solvent composition on the pH of buffered HPLC mobile phase and the pKa of analytes-a review. Sep Purif Rev 2007, 36: 231-255. 10.1080/15422110701539129View Article
- Wei Z, Na-na R, Wei-bo C, et al.: Determination of dissociation constant and oil/water partition coefficient of scutellarein. Journal of Yunnan University of Traditional Chinese Medicine 2010, 33(5):28-32.
- Wiczling P, Markuszewski MJ, et al.: Determination of pKa by pH gradient reversed-phase HPLC. Anal Chem 2004, 76(11):3069. 10.1021/ac049807qView Article
- Wiczling P, Kawczak P, Nasal A, et al.: Simultaneous determination of pKa and lipophilicity by gradient RP-HPLC. Anal Chem 2006, 78(1):239-249. 10.1021/ac0512103View Article
- Wiczling P, Waszczuk-Jankowska M, Markuszewski MJ, et al.: The application of gradient reversed-phase high-performance liquid chromatography to the pKa ang logkw determination of polyprotic analytes. J Chromatogr A 2008, 1214: 109-114. 10.1016/j.chroma.2008.10.085View Article
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.