HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) METHODS FOR AFRICAN POTATO AND EFAVIRENZ
4.3 Quantitative Analysis of Hypoxoside in African Potato Decoctions
Liquid bonded phase chromatography is the most widely-used because of its stability. The bonded phase columns have a limited sample capacity, therefore, small sample sizes have to be used to avoid column overload. There are two types of liquid bonded phase chromatography namely, normal phase and reversed phase. In normal phase chromatography, the stationary phase is polar and the MP is relatively non-polar, and is usually used to separate polar compounds. The retention times of polar compounds will be longer on normal phase chromatography. The most commonly-used type is reversed phase HPLC, where the stationary phase is non-polar relative to the MP performed using bonded octyl (C8) or octyldecyl group (C18) silane packings. The bonded phase packings are prepared by reaction of an organochlorosilane with a hydroxyl group of the silanol moiety. The organochlorosilanes often have a straight-chain octyl (C8) or octyldecyl group (C18).
4.3 Quantitative Analysis of Hypoxoside in African Potato Decoctions
4.3.1 Introduction
Reversed (RP) phase HPLC is a much-used analytical technique for the separation of chemical compounds in TM/CAM plant materials, as well as in pharmaceutical dosage forms.
As previously mentioned, this method is versatile and not influenced by the volatility or stability of the analyte, hence, it can be used to separate various classes of analytes including TMs/CAMs [203]. The versatility of HPLC has enabled the use of a variety of detectors which include ultraviolet-visible (UV-VIS), mass spectroscopy (MS), evaporative light scattering detection (ELSD), fluorescence, electrochemical and even nuclear magnetic resonance (NMR). HPLC is used for the comprehensive qualitative analysis (fingerprinting) of TMs/CAMs, and used to compare profiles because of its ability to provide useful content information [204, 205]. Compounds with strong chromophores that absorb light in the wavelength region of about 200 to 800 nm are usually monitored using UV-VIS detection.
Recent advances in technology have yielded a photodiode array (PDA) detector which is an extremely useful multi-wavelength detection technique. PDA detectors (also known as diode array detectors or DAD) can determine purity of each peak as well identify peaks with the aid of PDA spectral libraries. These features are very important in the analysis of TMs/CAMs, which usually contain many compounds, some of which may be present in only very small amounts. Compounds with differing electroactivity which may cause characteristic
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voltametric responses can be selectively measured using electrochemical detection (ECD).
Moreover, compounds that lack the necessary chromophores leading to poor UV-VIS can be detected by a refractive index (RI), or evaporative light scattering detector (ELSD) [206] as well as ECD.
Recent advances in mass spectrometric (MS) instrumentation have led to the incorporation of liquid chromatography coupled with mass spectroscopy (LC-MS-MS) for use in routine analysis [207]. This method allows for the identification and confirmation of the mass of compounds which can be of complex and diverse structures present in plant materials, and thus provides a powerful and useful application for both qualitative and quantitative analysis.
There is a dearth of information in the published literature describing the methods for the quantitative analysis of hypoxoside. A RP-HPLC method for the determination of hypoxoside distribution in three different Hypoxis species was reported by Vinesi et al in 1990 [208], however, no validation data and related information were provided.
In 1992, Betto et al [209] reported a RP-HPLC method using an acidic (pH 3.0) eluent to separate and determine some known norlignan glucosides isolated from Hypoxidaceae species. A gradient method was used, but here again, no validation data to support its application was provided, despite the claim that the method could be used for TMs and analysis of pharmaceutical preparations. Kruger et al [186] reported an HPLC method which was purported to be a quantitative analytical method for the analysis of hypoxoside and its metabolites in human plasma, urine and faeces. The MP consisted of guanidinine hydrochloride and ammonium sulphate for the sorption enrichment of xenobiotics in biological fluids. Validation and quantitative data of this method were also not reported, despite its application for the analysis of biological fluids from human subjects who had taken part in a clinical study. This method was also applied to extracts of H. rooperi and H.
latifolia for the identification of hypoxoside.
A validated RP-HPLC method used for the quantitative determination of hypoxoside in plant extracts including traditionally-prepared decoctions and in pharmaceutical dosage forms, has recently been reported by Nair et al [27]. The method used a binary mobile phase, detection was by PDA detector, and the method was characterised by high accuracy and precision.
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Therefore, this method was adopted for the analysis of traditionally-prepared decoctions of AP. Since this method was routinely used in our laboratory, a mini-validation comprising repeatability of the calibration plots by another analyst, was carried out.
4.3.2 Equipment and Reagents
The HPLC system consisted of a Waters Alliance system, model 2690, equipped with a pump, an autosampler, an online degasser, and a model 2995 PDA UV detector (Waters, Milford, MA, USA). Samples were centrifuged using an IEC HN-SII® Centrifuge (Damon/IEC Division, Needham Hts, Massachusetts, USA), and the hypoxoside standard (87.9%) was qualified for use and provided by a colleague, Dr. Patnala.
4.3.3 Chromatographic Conditions
Separation was achieved on a Phenomenex® Luna C18 (2) (5 µm, 150 mm × 4.6 mm i.d) column at 23 ± 2 °C, using acetonitrile: water (85:15 v/v) as the mobile phase. The flow rate of the mobile phase was 1 ml/min, 10 µl samples were injected, and hypoxoside elution was monitored by UV detection at λ = 260 nm. The internal standard used was sulphamerazine (SMZ).
4.3.4 Methods
The absorption spectrum of hypoxoside was determined by HPLC coupled with a DAD [27] . The purity of the standard provided was also assessed using the HPLC-DAD method, and the structure was confirmed using LCMS. Calibration plots were prepared using a range of calibration standards consisting of 5, 10, 20, 40 and 80 µg/ml.
4.3.5 Results and Discussion
The hypoxoside standard was readily soluble in methanol and water. The UV spectrum is depicted in Figure 4.3 below, and corresponds to the UV scan of hypoxoside previously reported [27, 186].
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AU
0.00 0.50 1.00 1.50 2.00 2.50
nm
220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
213.2
258.0
291.1
Figure 4.3. UV absorption spectrum of hypoxoside.
The calibration plots of hypoxoside were all linear using un-weighted regression analysis (Table 4.2).
Table 4.2. Linearity
DAY Y -intercept Gradient of calibration curve
Correlation coefficient
1 -0.021 0.031 0.999
2 -0.045 0.034 0.999
A typical chromatogram showing the separation of hypoxoside and the internal standard (SMZ) is shown in Figure 4.4.
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Figure 4.4. Chromatogram of calibration standard of hypoxoside with SMZ.
4.3.6 Conclusions
The data from the peak purity of hypoxoside using HPLC-UV confirmed the absence of UV absorbing impurities. The hypoxoside standard was thus sufficiently pure (87.9%) and was acceptable for use as an analytical reference for quantitative analysis.
4.4 DEVELOPMENT AND VALIDATION OF A HPLC METHOD FOR THE