ตำรามาตรฐานยาสมุนไพรไทย
Thai Herbal Pharmacopoeia
สำนักยาและวัตถุเสพติด กรมวิทยาศาสตร์การแพทย์ กระทรวงสาธารณสุข
Bureau of Drug and Narcotic, Department of Medical Sciences, Ministry of Public Health(Tinospora crispa (L.) Hook.f. & Thomson)
(Nelumbo nucifera Gaertn.)
(Centella asiatica (L.) Urb.)
(Centella Dry Extract)
(Centella Cream)
(Mesua ferrea L.)
(Piper sarmentosum Roxb.)
(Piper sarmentosum Roxb.)
(Pterocarpus santalinus L. f.)
(Santalum album L.)
(Senna tora (L.) Roxb.)
(Senna alata (L.) Roxb.)
(Senna Alata Tea)
(Piper retrofractum Vahl)
(Myristica fragrans Houtt)
(Andrographis paniculata (Burm. f.) Nees)
(Andrographis Capsules)
(Allium ascalonicum L.)
(Ocimum tenuiflorum L.)
(Curcuma longa L.)
(Turmeric Capsules)
(Turmeric Dry Extract)
(Turmeric Dry Extract Capsules)
(Arcangelisia flava (L.) Merr.)
(Curcuma sp.)
Harrisonia perforata (Blanco) Merr.
(Aristolochia pierrei Lecomte)
(Zingiber officinale Roscoe)
(Ginger Capsules)
(Ginger Tea)
(Cassia fistula L.)
(Nardostachys jatamansi (D. Don) DC.)
(Angelica sinensis (Oliv.) Diels)
Artemisia annua L.
(Ligusticum sinense Oliv. cv. Chuanxiong)
(Neopicrorhiza scrophulariiflora Pennell)
(Atractylodes lancea (Thunb.) DC.)
(Aucklandia lappa Decne)
(Terminalia chebula Retz.)
(Angelica dahurica (Hoffm.) Benth. & Hook. f. ex Franch. & Sav. var. dahurica)
(Kaempferia parviflora Wall. ex Baker)
(Hibiscus sabdariffa L.)
(Roselle Tea)
(Allium sativum L.)
(Zingiber zerumbet (L.) Sm.)
(Wurfbainia testacea (Ridl.) Škorničk.& A. D. Poulsen)
(Cannabis sativa L.)
(Myristica fragrans Houtt)
(Dracaena cochinchinensis (Lour.) S. C. Chen)
(Ficus racemosa L.)
(Hyptis suaveolens (L.) Poit.)
Clerodendrum indicum (L.) Kuntze
(Phyllanthus emblica L.)
(Citrus hystrix DC.)
(Citrus hystrix DC.)
(Areca catechu L.)
(Momordica charantia L.)
Moringa oleifera Lam.
(Aegle marmelos (L.) Corrêa)
(Solanum trilobatum L.)
(Morus alba L.)
Gynostemma pentaphyllum(Thunb.)
Makino
(Clinacanthus nutans (Burm. f.) Lindau)
(Cissus quadrangularis L.)
(Mimusops elengi L.)
(Zingiber montanum (J. König) Link. ex A. Dietr.)
(Piper betle L.)
(Capsicum annuum L.)
(Capsicum Oleoresin)
(Capsicum Gel)
(Piper nigrum L.)
(Piper nigrum L.)
(Eurycoma longifolia Jack)
(Thunbergia laurifolia Lindl.)
(Piper wallichii (Miq.) Hand.-Mazz.)
Senna garrettiana (Craib) H. S. Irwin & Barneby
(Terminalia bellirica (Gaertn.) Roxb.)
(Terminalia chebula Retz.)
(Caesalpinia bonduc (L.) H. Roxb.)
(Tarlmounia elliptica (DC.) H. Rob., S. C. Keeley, Skvaria & R. Chan)
(Hog Creeper Vine Dry Extract Capsiles)
(Hog Creeper Vine Dry Extract)
(Brachypterum scandens (Roxb.) Miq.)
(Lepidium sativum L.)
(Nigella sativa L.)
(Cuminum cyminum L.)
(Foeniculum vulgare Mill.)
(Plantago ovata Forssk.)
(Pimpinella anisum L.)
(Carum carvi L.)
(Anethum graveolens L.)
(Trachyspermum ammi (L.) Sprague)
Albizia procera (Roxb.) Benth.
(Acorus calamus L.)
(Tiliacora triandra (Colebr.) Diels)
Cyanthillium cinereum (L.) H. Rob.
(Orthosiphon aristatus (Blume) Miq.)
Murdannia loriformis (Hassk.) R. S. Rao & Kammathy
(Capparis micracantha DC.)
(Chrysopogon zizanioides (L.) Roberty)
(Cyperus rotundus L.)
(Cannabis sativa L.)
(Syzygium aromaticum (L.) Merr. & L. M. Perry)
(Boesenbergia rotunda (L.) Mansf.)
(Acanthus ebracteatus Vahl)
(Acanthus ilicifolius L.)
(Kaempferia galanga L.)
(Curcuma comosa Roxb.)
Betula alnoides Buch.-Ham. ex D. Don
Cannabis sativa L.
Carthamus tinctorius L
Mitragyna speciosa (Korth.) Havil
Mallotus repandus (Rottler) Müll. Arg
Azadirachta indica A. Juss. var. siamensis Valeton
Azadirachta indica A. Juss. var. siamensis Valeton
Punica granatum L.
Rhinacanthus nasutus (L.) Kurz
Baliospermum solanifolium (Burm.) Suresh
Curcuma aeruginosa Roxb
Boesenbergia kingii Mood & L. M. Prince
Senegalia rugata (Lam.) Britton & Rose
Acacia concinna (Willd.) DC.
Senegalia rugata (Lam.) Britton & Rose
Acacia concinna (Willd.) DC.
Senna alexandriana Mill. var. alexandriana
Cassia acutifolia Delile, Cassia angustifolia Vahl
Butea superba Roxb. ex Willd.
[Plaso superba (Roxb. ex Willd.) Kuntze, Rudolphia superba (Roxb. ex Willd.) Poir.
Pueraria candollei Graham
ex Benth. var. mirifica (Airy Shaw & Suvat.) Niyomdham
Streblus asper Lour.
Suregada multiflora (A. Juss.) Baill. (Gelonium
multiflorum A. Juss.
The term Liquid chromatography (LC), in this Pharmacopoeia, is referred to as HPLC, both high-pressure and high-performance. It is a separation technique based on a solid stationary phase and a liquid mobile phase. Separations are achieved by partition (mass distribution), adsorption, ion-exchange, or stereochemical-interaction processes, depending upon the type of stationary phase used. Unless otherwise specified, all information below is valid for LC as well as for LC using reduced particle-size columns (e.g., sub-2 μm). The latter requires instrumentation characterized by the capability to apply higher pressures (typically up to 100 MPa, i.e., about 15,000 psi), lower extra-column band broadening, improved gradient mixing and a higher sampling rate in the detection system.
Apparatus
A liquid chromatograph consists of a reservoir containing the mobile phase, a pump to force the mobile phase through the system at high pressure, an injector to introduce the sample into the mobile phase, a chromatographic column, a detector, and a data acquisition system.
PUMPING SYSTEMS LC pumping systems deliver metered amounts of mobile phase from the solvent reservoirs to the column though high-pressure tubing and fittings. Microprocessor-controlled pumping systems are capable of accurately delivering a mobile phase of either constant (isocratic elution) or varying composition (gradient elution), according to a defined programme. In the case of gradient elution, pumping systems which deliver solvent(s) from several reservoirs are available and solvent mixing can be achieved on either the low- or high-pressure side of the pump(s).
INJECTORS After dissolution in mobile phase or other suitable solution, compounds to be chromatographed are injected into the mobile phase. Fixed-loop and variable volume devices operated manually or by an auto-sampler are used.
STATIONARY PHASES For most pharmaceutical analyses, separation is achieved by partition of compounds in the test solution between the mobile phase and the stationary phase. A system consisting of a polar stationary phase and a nonpolar mobile phase is described as normal-phase chromatography, while the opposite arrangement, a polar mobile phase and a nonpolar stationary phase, is called reversed-phase chromatography. There are many types of stationary phase materials employed in LC, including:
— silica, alumina or porous graphite, used in normal-phase chromatography, where the separation is based on differnces in adsorption and/or mass distribution(partition chromatography),
— resins or polymers with acid or basic groups, used in ion-exchange chromatography, where separation is based on competition between the ions to be separated and those in the mobile phase,
— porous silica or polymers, used in size-exclusion chromatography, where separation is based on differences between the volumes of the molecules, corresponding to steric exclusion,
— a variety of chemically modified supports prepared from polymers, silica or porous graphite, used in normal-phase (adsorption chromatography) and reversed-phase LC, where the separation is based principally on partition of the molecules between the mobile phase and the stationary phase,
— special chemically modified stationary phases, e.g., cellulose or amylose derivatives, proteins or peptides, cyclodextrins, etc., for the separation of enantiomers (chromatography on chiral stationary phases).
Most separations are based upon partition mechanisms utillzing chemically modified silica as the stationary phase and polar solvents as the mobile phase. The surface of the support, e.g., the silanol groups of silica, is reacted with various silane reagents to produce covalently bound silyl derivatives covering a varying number of active sites on the surface of the support. The nature of the bonded phase is an important parameter for determining the separation properties of the chromatographic system.
Commonly used bonded phases are shown below:
octyl (C8) | = | Si-[CH2]7-CH3 |
octadecyl (C18) | = | Si-[CH2]17-CH3 |
phenyl (C6H5) | = | Si-[CH2]n-C6H5 |
cyanopropyl (CN) | = | Si-[CH2]3-CN |
aminopropyl (NH2) | = | Si-[CH2]3-NH2 |
diol | = | Si-[CH2]3-O-CH(OH)-CH2-OH |
Unless otherwise stated by the manufacturer, silica-based reversed-phase columns are considered to be stable in mobile phases having an apparent pH in the range of 2.0 to 8.0. Columns containing porous graphite or particles of polymeric materials such as styrenedivinylbenzene copolymer are stable over a wider pH range. Analysis using normal-phase chromatography with unmodified silica, porous graphite or polar chemically modified silica, e.g.,cyanopropyl or diol, as the stationary phase with a nonpolar mobile phase is applicable in certain cases. For analytical separations, the particle size of the most commonly used stationary phases varies between 2 μm and 10 μm. The particles may be spherical or irregular, of varying porosity and specific surface area. These properties contribute to the chromatographic behaviour of a particular stationary phase. In the case of reversed phases, the nature of the stationary phase, the extent of bonding, e.g., expressed as the carbon loading, and whether the stationary phase is end-capped (i.e., part of the residual silanol groups being silylated) are additional determining factors. Tailing of peaks, particularly of basic substances, can occur when residual silanol groups are present. In addition to porous particles, superficially porous or monolithic materials may be used.
COLUMNS Columns, made of stainless steel, lined stainless steel, and polymers of varying length and internal diameter are used for analytical chromatography. Columns with internal diameters of less than 2 mm are often referred to as microbore columns. The temperature of the mobile phase and the column must be kept constant during an analysis. Most separations are performed at room temperature, but some require a different temperature for optimal performance.
In LC procedures, a guard column may be used with the following requirements, unless otherwise indicated in the individual monograph: (a) the length of the guard column must be not more than 15 per cent of the length of the analytical column, (b) the internal diameter must be the same or smaller than that of the analytical column, and (c) the packing material should be the same as the analytical column and contain the same bonded phase. In any case, all system suitability requirements specified in the offcial procedure must be met with the guard column installed.
MOBILE PHASES
For normal-phase chromatography, less polar organic solvents are generally employed. The residual water content of the solvents used in the mobile phase is to be strictly controlled to obtain reproducible results. In reversed-phase LC, aqueous phases, with or without organic solvents, are employed.
Components of the mobile phase are usually filtered to remove particles greater than 0.45 μm (or 0.2 μm when the stationary phase is made of sub-2 μm particles and when special detectors, e.g., light scattering detectors, are used). Solvents are normally degassed before pumping by sparging with helium, sonication and/or using on-line membrane/ vacuum modules to avoid the creation of gas bubbles in the detector cell. Solvents for the preparation of the mobile phase are normally free of stabilizers and, if an ultraviolet detector is employed, are transparent at the wavelength of detection. Solvents and other components employed are to be of appropriate quality. Adjustment of the pH, if necessary, is effected using only the aqueous componnent of the mobile phase and not the mixture. If buffer solutions or saline solutions are used, adquate rinsing of the system is carried out with a 5 per cent v/v mixture of the organic part of the mobile phase in water to prevent crystallization of salts after completion of the analysis. Mobile phases may contain other components, e.g., a counter-ion for ion-pair chromatography or a chiral selector for chromatography using an achiral stationary phase.
DETECTORS
Ultraviolet/visible (UV/Vis) spectrophotometers, including diode array detectors, are the most commonly employed detectors. Fluorescence spectrophotometers, differential refractometers (RI), electrochemical detectors (ECD), light scattering detectors, charged aerosol detectors (CAD), mass spectrometers (MS), radioactivity detectors or other special detectors may be used.
Procedure
Criteria for assessing the suitability of the system are described in the “Chromatographic Separation Techniques” (Appendix 3.9). The extent to which adjustments of parameters of the chromatographic system can be made to satisfy the criteria of system suitability are also given in this Appendix.
In general, the steps involved in the liquid chromatographic technique are as follows:
1. Equilibrate the column and detector with the mobile phase at the specified flow rate until a stable baseline is achieved.
2. Prepare the sample and the standard solutions. The solutions must be free from solid particles.
3. Inject the standard/sample solution through the injector, or use an autosampler.
4. Perform the isocratic/gradient programme.
5. Record the chromatogram.
6. Analyze as specified in the monograph.
In quantitative work, particularly where the use of an internal standard is not specified in the monograph, the use of a fixed-volume loop injector is recommended. In certain exceptional cases the use of peak heights alone is prescribed in the monograph; where this is the case peak heights should be used irrespective of the symmetry factor. The column is usually made of stainless steel and its dimensions are stated in the monograph as length × internal diameter. The nominal diameter of the particles of the stationary phase is stated in parentheses following the name of stationary phase prescribed in the monograph. In most cases reference is made to a particular commercial brand that has been found to be suitable for the purpose, but such statements do not imply that a different but equivalent commercial brand may not be used. The separation should be carried out at a constant room temperature unless otherwise specified in the monograph. When using mobile phases of high pH with a silica-based column, it is advisable to use a pre-column before the analytical column.
Unless otherwise specified in the monograph, the detector consists of a photometric detector fitted with a low-volume flow cell(about 10 μL is suitable); the wavelength setting is specified in the monograph.