A simple method for HPLC retention time prediction: linear calibration using two reference substances.

BACKGROUND: Analysis of related substances in pharmaceutical chemicals and multi-components in traditional Chinese medicines needs bulk of reference substances to identify the chromatographic peaks accurately. But the reference substances are costly. Thus, the relative retention (RR) method has been widely adopted in pharmacopoeias and literatures for characterizing HPLC behaviors of those reference substances unavailable. The problem is it is difficult to reproduce the RR on different columns due to the error between measured retention time (tR) and predicted tR in some cases. Therefore, it is useful to develop an alternative and simple method for prediction of tR accurately. METHODS: In the present study, based on the thermodynamic theory of HPLC, a method named linear calibration using two reference substances (LCTRS) was proposed. The method includes three steps, procedure of two points prediction, procedure of validation by multiple points regression and sequential matching. The tR of compounds on a HPLC column can be calculated by standard retention time and linear relationship. RESULTS: The method was validated in two medicines on 30 columns. CONCLUSION: It was demonstrated that, LCTRS method is simple, but more accurate and more robust on different HPLC columns than RR method. Hence quality standards using LCTRS method are easy to reproduce in different laboratories with lower cost of reference substances.

Combination of quantitative analysis and chemometric analysis for the quality evaluation of three different frankincenses by ultra high performance liquid chromatography and quadrupole time of flight mass spectrometry.

Frankincense has gained increasing attention in the pharmaceutical industry because of its pharmacologically active components such as boswellic acids. However, the identity and overall quality evaluation of three different frankincense species in different Pharmacopeias and the literature have less been reported. In this paper, quantitative analysis and chemometric evaluation were established and applied for the quality control of frankincense. Meanwhile, quantitative and chemometric analysis could be conducted under the same analytical conditions. In total 55 samples from four habitats (three species) of frankincense were collected and six boswellic acids were chosen for quantitative analysis. Chemometric analyses such as similarity analysis, hierarchical cluster analysis, and principal component analysis were used to identify frankincense of three species to reveal the correlation between its components and species. In addition, 12 chromatographic peaks have been tentatively identified explored by reference substances and quadrupole time-of-flight mass spectrometry. The results indicated that the total boswellic acid profiles of three species of frankincense are similar and their fingerprints can be used to differentiate between them.

Evaluation of traditional Chinese herbal medicine: Chaihu (Bupleuri Radix) by both high-performance liquid chromatographic and high-performance thin-layer chromatographic fingerprint and chemometric analysis.

Chaihu (Bupleuri Radix), roots of Bupleurum chinense and B. scorzonerifolium, is an authentic Chinese Materia Medica in the Chinese Pharmacopoeia. Some other species such as the roots of B. falcatum, B.bicaule and B. marginatum var. stenophyllum similar to Chaihu can also be occasionally found in local raw herb markets. The quality of 33 lots of authenticated Chaihu samples vs. 31 lots of commercial samples was evaluated by both high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) and high-performance thin-layer chromatography (HPTLC) analyses of its principal bioactive components (saikosaponins). The pre-treated data acquired from both HPLC fingerprints and HPTLC fluorescent images were processed by chemometrics for similarity and pattern recognition, including Artificial Neural Networks (ANNs), k-nearest neighbor (k-NN) and an expert's panel. It was apparent that k-NN classifier exhibited good performance with sufficient flexibility for processing HPTLC fingerprint images which were otherwise not easily dealt with by other algorithms due to the shift of R(f) values and varying hue/saturation of the band colours between different TLC plates. These two chromatographic fingerprint methods can be considered complementary measure of quality control. The roots of Chaihu from different species of the genus Bupleurum could readily be distinguished from each other so that commercial samples can easily be classified. Chaihu collected from several major herbal distribution centers was found to belong to B. chinense with great variation in the content of its major saikosaponins.

[Processing method and HPLC fingerprint of charred radix Scutellariae].

OBJECTIVE: To investigate the process and HPLC fingerprint of Charred Radix Scutellariae, and lay a foundation of Charred Radix Scutellariae quanlity control mode. METHOD: Select HPLC-UV fingerprint. Chromatogram condition: Hypersil C18 column (5.0 mm x 200 mm, 5 microm), mixtures of methanol, 0.4% phosphoric acid and acetonitrile as mobile phase in a gradient mode. Flow rate: 1.0 mL x min; Detection wavelength was set at 277 nm. RESULT: There were no evident differences among Charred Radix Scutellariae that were normatively manufactured and processed. CONCLUSION: This test proves the process is feasible, this method recurs well and can be used to provide scieitific basis for the normative process and quanlity control mode of Charred Radix Scutellariae.

[Study of HPLC-DAD fingerprint on complex traditional Chinese medicine proprietary preparation-Baoji pills].

OBJECTIVE: Based on 'Back-tracking' method, identification and quality evaluation of complex traditional Chinese medicine (TCM) preparation of Baoji pills (BJP) were carried out by HPLC fingerprint analysis. METHOD: HPLC-DAD fingerprint of BJP was conducted with Zorbax SB-C18 column and non-linear elution with the mobile phase consisted of acetonitrile-0.5% glacial acetic acid at column temperature 30 degrees C and detective wavelengths of 250 nm and 283 nm. From the established chromatographic pattern of BJP, track backward to the corresponding crude herbal drugs in the formula, attribution ofmost peaks in the BJP fingerprint can be disclosed. RESULT: The BJP HPLC fingerprint consisted of 44 peaks among which 35 peaks were assigned by parallel comparison with the fingerprint of the 10 corresponding crude drugs in the formula such as pueraria, pummelo peel, and magnolia bark, etc. and 22 peaks we reidentified by comparison with the chemical reference substances. CONCLUSION: The established HPLC fingerprint represents the whole character of BJP, which enhanced the specialty for control and assessment of the product quality. It exemplified much more effective for quality control than selecting any marker for qualitative or quantitative testing target. And the Back-tracking' experimental method extended the study mentality for complex formula TCM products chromatographic fingerprinting analysis.

[HPLC fingerprinting of radix paeoniae alba].

To establish a sensitive and specific HPLC method for quality control of Radix Paeoniae Alba, HPLC method was applied for quality assessment of Radix Paeoniae Alba. HPLC analysis was performed on a Symmetry C18 column (250 mm x 4. 6 mm ID, 5 microm, Waters, USA). The mobile phase consisted of acetonitrile (solvent A) and water containing 0.1% (v/v) phosphoric acid (solvent B) at a constant flow rate of 0.8 mL x min(-1). An increasing linear gradient (v/v) of solvent A was used (t/min, % A): (0,10), (5,10), (25,15), (45, 22), (46, 65), (50, 80) and (60, 80). The column temperature was set at 25 degrees C. The chromatograms were monitored at 230 nm and the on-line UV spectra were recorded in the range of 190 - 400 nm. The HPLC chromatographic fingerprinting of Radix Paeoniae Alba, showing 11 characteristic peaks, was established from 28 lots of Radix Paeoniae Alba. The areas of main chromatographic peaks were found to complied with the following rule: paeoniflorin > 1, 2, 3, 4, 6-penta-O-galloyl-glucos > albiflorin > methyl gallate > other compounds. The chromatographic fingerprinting of Radix Paeoniae Alba with high specificity can be used to control its quality and assure lot-to-lot consistency.

High-performance thin-layer chromatographic fingerprints of isoflavonoids for distinguishing between Radix Puerariae Lobate and Radix Puerariae Thomsonii.

The roots of Pueraria lobata (Wild.) Ohwi and Pueraria thomsonii Benth have been officially recorded in all editions of Chinese Pharmacopoeia under the same monograph 'Gegen' (Radix Puerariae, RP). However, in its 2005 edition, the two species were separated into both individual monographs, namely 'Gegen' (Radix Puerariae Lobatae, RPL) and 'Fenge' (Radix Puerariae Thomsonii, RPT), respectively, due to their obvious content discrepancy of puerarin, the major active constituent. In present paper, the fingerprint of high-performance thin-layer chromatography (HPTLC) combining digital scanning profiling was developed to identify and distinguish the both species in detail. The unique properties of the HPTLC fingerprints were validated by analyzing ten batches of Pueraria lobata and P. thomsonii samples, respectively. The common pattern of the HPTLC images of the roots of Pueraria spp. and the respective different ratios of the chemical distribution can directly discern the two species. The corresponding digital scanning profiles provided an easy way for quantifiable comparison among the samples. Obvious difference in ingredient content and HPTLC patterns of the two species questioned their bio-equivalence and explained that recording both species separately in the current edition of Chinese Pharmacopoeia (2005 edition) is reasonable due to not only the content of major constituent, puerarin, but also the peak-to-peak distribution in the fingerprint and integration value of the total components. Furthermore, the HPTLC fingerprint is also suitable for rapid and simple authentication and comparison of the subtle difference among samples with identical plant resource but different geographic locations.