The component herbs in the formula are as follows: Pogostemon cablin (herb) (4.5%, w/w), Angelica dahurica (root) (2%), Artemisia scoparia (herb) (13%), Atractylodes macrocephala (rhizome)(9%), Aucklandia lappa (root) (3%), Bupleurum chinense (root) (4.5%), Citrus reticulate (fruit peel) (3%), Codonopsis pilosula (root) (7%), Coix lacryma-jobi (seed) (7%), Coptis chinensis (rhizome) (3%), Fraxinus rhynchophylla (bark) (4.5%), Glycyrrhiza uralensis (root) (4.5%), Magnolia officinalis (bark) (4.5%), Paeonia lactiflora (root)(3%), Plantago asiatica (seed) (4.5%), Phellodendron amurense (bark) (4.5%), Poria cocos (fruiting body) (4.5%), Saposhnikovia divaricata (root) (3%), Schisandra chinensis (fruit) (7%) and Zingiber officinale (rhizome) (4.5%). All 20 herbs were acquired in the prescribed proportions (% w/w) from Zhixin Chinese Pharmaceutical Co. Ltd. (Guangzhou, China). The aggregate weight of the 20 herbs was 400 kg (Additional File 1). Voucher samples (#IBS-01 to IBS-20) were deposited at the herbarium of the School of Chinese Medicine, Chinese University of Hong Kong (Hong Kong SAR, China). The individual bulk herb samples were stored in air-tight containers kept in air-conditioned environment until use. The herbs were identified in both Chinese and botanical (Latin binomial) names. When two or more species share the same Chinese name, only one species was selected for chemical and biological/clinical studies.
All 20 herbs were authenticated macroscopically and microscopically. Macroscopic examinations included measurements of appearance, size, shape, color, texture, odor, taste, fracture and other characteristics of a herb according to pharmacopoeias [10–13]. Microscopic examinations determined characteristic elements of each herb in both tissue and powder forms. In cross sectional examination, herbal material was softened by immersion in water, alcohol or glycerin prior to sectioning. Sliced tissue, prepared with a microtome, was mounted on a glass microscope slide and clarified with chloral hydrate, lactochloral and/or sodium hypochlorite, followed by phloroglucinol, potassium iodide or Sudan Red. In powder analysis, each herbal material was pulverized to 65-mesh in size, mounted on a microscope slide, cleared with chloral hydrate, lactochloral and/or sodium hypochlorite, and then examined for the presence, size, shape and numbers of characteristic elements and inclusions such as vessels, calcium crystals, crystalline fibers, stone cells and starch grains. The examination protocols followed the World Health Organization (WHO) Quality Control Methods for Medicinal Plant Materials , the Pharmacopoeia of the People's Republic of China (CP)  and the Hong Kong Chinese Materia Medica Standards (HKCMMS) [11–13]. The recorded macroscopic and microscopic data for each herb were verified against those in the CP and/or the HKCMMS, coupled with visual comparison with available reference samples.
Reference marker compounds and reagents
Reference marker compounds for qualitative and quantitative high performance liquid chromatography (HPLC) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) and further validated by mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) and purity (>98%) analysis with HPLC and/or liquid chromatography-mass spectrometry (LC-MS). Chemicals and general solvents were of reagent grade and HPLC solvents were of HPLC grade (BDH, United Kingdom).
Purity and contaminant determination
Purity rubric tests, including foreign matters, total ash, acid-insoluble ash, water and extractive contents, were carried out according to the CP or HKCMMS [10–13]. Determination of heavy metals (arsenic, cadmium, mercury, and lead), pesticides, microbials and microbial toxin (aflatoxin) was carried out according to the HKCMMS [11–13]. Briefly, for heavy metal analysis, the herbal matrix was dissolved by microwave-assisted acid digestion, and the presence and quantity of mercury, lead, arsenic, and/or cadmium, if any, were determined by inductively coupled plasma-mass spectrometry (ICP-MS). Pesticide residues (e.g., aldrin, dieldrin, chlordane, dichlorodiphenyltrichloroethane, endrin, heptachlor, hexachlorobenzene, hexachlorocyclohexane isomers, lindane and quintozene) were quantitatively determined with gas chromatography (GS). Mycotoxins (aflatoxins B1, B2, G1 and G2) were detected as previously described [11–13, 15]. Microorganism examinations included total bacteria, mould and yeast, Escherichia coli and Salmonella counts as described in the CP .
Chromatographic fingerprint analysis HPLC fingerprinting with one or more reference markers was carried out according to the HKCMMS and/or CP [10–13]. As an example, the procedure used for Rhizoma Coptidis (Huanglian) is described here. The herb was ground to powder, extracted in MeOH by ultrasonication for 30 minutes and filtered. The chromatographic system consisted of an Agilent 1100 HPLC system (Agilent Technologies, USA) equipped with a secondary pump, a diode-array detector, an autosampler, and a column compartment, an Alltech Alltima C18 column (4.6 × 250 mm) (Alltech, USA) packed with 5 μm diameter particles and an Alltech Alltima guard column (7.5 × 4.6 mm, 5 μm) (Alltech, USA); solvent system: 0.1% trifluoroacetic acid (%, v/v) (A) and acetonitrile (%, v/v) (B) with a linear gradient elution, 0% B-50% B at 0-48 minutes, 50% B-100% B at 48-55 minutes, 100% B was held for five minutes; flow rate: 1.0 ml/min; detection: 346 nm; reference marker: berberine. Information of the reference marker compounds for each herb is available in Additional File 1.
Quantitative determination of selected marker compound(s) in each herb was performed with HPLC analysis. As an example, the quantitative analysis of Cortex Magnoliae Officinalis (Houpo) is described here. Preparation of the herb and the HPLC setup were the same as described above. The mobile phase contained 0.4% formic acid and acetonitrile (35:65); flow rate: 1 ml/min; detection: 294 nm. Information of the reference marker compounds for each herb is available in Additional File 1.
Inter-laboratory methods validation
Inter-laboratory validation of fingerprint and quantitative HPLC analytical protocols were carried out in laboratories at the Chinese University of Hong Kong and the University of Western Sydney prior to use. Both laboratories followed the identical experimental protocols and the results were critically compared.
Production of herbal extracts and the IBS-20 formula
The 20 dried herbs were individually reduced in size by milling or slicing and mixed in the prescribed proportion (% w/w), followed by extraction with water under GMP at the Hong Kong Institute of Biotechnology (Hong Kong Special Administrative Region, China). Briefly, the herb mixture (400 kg) was decocted with 10-fold (w/v) of boiling distilled water for 60 minutes, cooled and collected. After fresh boiling water was added, the mixture was decocted for a second time. The cooled extracts were pooled, filtered, concentrated and spray dried to obtain a powder (34% yield w/w based on raw herbs). An aliquot was set aside for chemical and pre-clinical biological studies. The remaining powdered extract was then formulated with water-soluble starch (excipient) in a ratio of 1:1 into the clinical product in the form of an aluminum foil packed sachet.
Chemical standardization of the IBS-20 formula
Chemical standardization of the clinical herbal extract with selected reference markers was performed with HPLC coupled with diode array detection and atmospheric pressure chemical ionization mass spectrometry (HPLC-DAD-APCIMS) analysis. Briefly, the powdered extract was sonicated in MeOH for 30 minutes and filtered through a cellulose syringe filter. An aliquot (10 μl) of filtrate was injected into an Agilent 1100 HPLC system equipped with an Alltech Alltima C18 column (4.6 × 250 mm, 5 μm diameter). The mobile phase consisted of 0.1% trifluoroacetic acid in water (v/v) (A) and acetonitrile (B) with the gradient elution conditions as follows: 12% B-13% B at 0-10 minutes, 13% B - 16% B at 10-40 minutes, 16% B-36.4% B at 40-67 minutes, 36.4% B-60% B at 67-100 minutes, from 100-120 minutes a gradient was applied to 100% B and was held for five minutes, followed by a 10-minute equilibration period at 12% B; flow rate: 1.5 ml/min; temperature: 27.5°C (constant). For detection, an Agilent 1100 series LC/MSD trap (Agilent Technologies, USA) was connected to the HPLC system via an APCIMS interface. Ultra-high purity helium was used as the collision gas and high purity nitrogen as the nebulizing gas. The optimized parameters in the positive ionization mode were as follows: nebulizer gas pressure: 50 psi; dry gas flow: 5.0 l/min; dry temperature: 350°C; vaporizer temperature: 400°C; full-scan MS analysis in the range of m/z 100-2200. The reference marker compounds present in the sample were identified by retention time, MS fragmentation and UV spectra.
The stability of the chemically standardized herbal formula was monitored in real time over a period of at least one year. The HPLC fingerprint profiles as well as the quantitative content of eight selected major marker compounds in the clinical formula were measured on days 0, 2, 3, 7, 14, 30, 60, 90, 180, 360, 450 post-production with HPLC and LC-MS respectively.