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EFFICACY: Quality

III. QUALITY

The proof of quality has been described in the Pharmacopoea Austriaca VIII, European Pharmacopoeia and in the Deutsches Arzneibuch (German Pharmacopoeia), 6th edition.

Greater celandine - Extract from the European Pharmacopoeia 6.0
6.0/1861
Greater celandine
Chelidonii herba
DEFINITION
Dried, whole or cut aerial parts of Chelidonium majus L. collected during flowering.
Content: minimum 0.6 per cent of total alkaloids, expressed as chelidonine (C20H19NO5; Mr 353.4) (dried drug).
CHARACTERS
Macroscopic and microscopic characters described under identification tests A and B.
IDENTIFICATION
A. The stems are rounded, ribbed, yellowish to greenish-brown, somewhat pubescent, about 3 mm to 7 mm in diameter, hollow and mostly collapsed. The leaves are thin, irregularly pinnate, the leaflets ovate to oblong with coarsely dentate margins, the terminal leaflet often three-lobed; the adaxial surface is bluish-green and glabrous, the abaxial surface paler and pubescent, especially on the veins. The flowers have 2 deeply concavo-convex sepals, readily removed, and 4 yellow, broadly ovate, spreading petals about 8 mm to 10 mm long; the stamens are numerous, yellow, and a short style arises from a superior ovary; long, capsular, immature fruits are rarely present.
B. Reduce to a powder (355). The powder is dark greyish-green to brownish-green. Examine under a microscope using chloral hydrate solution R. The powder shows the following diagnostic characters: numerous fragments of leaves in surface view, the epidermal cells with sinuous walls; anomocytic stomata (2.8.3) occur on the abaxial surface only; covering trichomes long, uniseriate, with thin walls and usually fragmented; vascular tissue from the leaves and stems with groups of fibres, pitted and spirally thickened vessels and associated latex tubes with yellowish-brown contents; occasional fragments of the corolla with thin-walled, partly papillose cells containing numerous pale yellow droplets of oil; spherical pollen grains about 30 µm to 40 µm in diameter with 3 pores and a finely pitted exine.
C. Thin-layer chromatography (2.2.27).
Test solution. To 0.4 g of the powdered drug (710) add 50 ml of dilute acetic acid R. Boil the mixture under a reflux condenser in a water-bath for 30 min. Cool and filter. To the filtrate add concentrated ammonia R until a strong alkaline reaction is produced. Shake with 30 ml of methylene chloride R. Dry the organic layer over anhydrous sodium sulphate R, filter and evaporate in vacuo to dryness. Dissolve the residue in 1.0 ml of methanol R.
Reference solution. Dissolve 2 mg of papaverine hydrochloride R and 2 mg of methyl red R in 10 ml of alcohol R.
Plate: TLC silica gel plate R.
Mobile phase: anhydrous formic acid R, water R, propanol R (1:9:90 V/V/V).
Application: 10 µl as bands.
Development: over a path of 10 cm.
Drying: in air.
Detection: spray with potassium iodobismuthate solution R and dry the plate in air; spray with sodium nitrite solution R and allow the plate to dry in air; examine in daylight.
Results: see below the sequence of the zones present in the chromatograms obtained with the reference solution and the test solution. Furthermore, other weaker zones may be present in the chromatogram obtained with the test solution.

Top of the plate

_______

_______

Methyl red: a red zone

A brown zone

 

A brown zone

Papaverine: a greyish-brown zone

A greyish-brown zone

_______

_______

 

 

 

A brown zone

 

A brown zone

Reference solution

Test solution

TESTS
Foreign matter (2.8.2): maximum 10 per cent.
Loss on drying (2.2.32): maximum 10.0 per cent, determined on 1.000 g of the powdered drug (355) by drying in an oven at 100-105 °C for 2 h.
Total ash (2.4.16): maximum 13.0 per cent.
ASSAY
Test solution. To 0.750 g of the powdered drug (710), add 200 ml of dilute acetic acid R and heat on a water-bath for 30 min, shaking frequently. Cool and dilute to 250.0 ml with dilute acetic acid R. Filter. Discard the first 20 ml of the filtrate. To 30.0 ml of the filtrate add 6.0 ml of concentrated ammonia R and 100.0 ml of methylene chloride R. Shake for 30 min. Separate the organic layer, place 50.0 ml in a 100 ml round-bottomed flask and evaporate to dryness in vacuo at a temperature not exceeding 40 °C. Dissolve the residue in about 2-3 ml of alcohol R, warming slightly. Transfer the solution to a 25 ml volumetric flask by rinsing the round-bottomed flask with dilute sulphuric acid R and dilute to 25.0 ml with the same solvent. To 5.0 ml of the solution, add 5.0 ml of a 10 g/l solution of chromotropic acid, sodium salt R in sulphuric acid R in a 25 ml volumetric flask, stopper the flask and mix carefully. Dilute to 25.0 ml with sulphuric acid R and stopper the flask.
Compensation solution. At the same time and in the same manner, place in a 25 ml volumetric flask 5.0 ml of dilute sulphuric acid R and 5.0 ml of a 10 g/l solution of chromotropic acid, sodium salt R in sulphuric acid R, stopper the flask and mix carefully. Dilute to 25.0 ml with sulphuric acid R and stopper the flask.
Place both solutions on a water-bath for 10 min. Cool to about 20 °C and dilute if necessary to 25.0 ml with sulphuric acid R. Measure the absorbance (2.2.25) of the test solution at 570 nm.
Calculate the percentage content of total alkaloids, expressed as chelidonine, from the expression:

A x 2.23
m

i.e. taking the specific absorbance of chelidonine to be 933.

A

absorbance at 570 nm,

m

mass of the substance to be examined, in grams.

 

Thiotepa – Extract from the US Pharmacopoeia, 24th edition

„Thiotepa

C6H12N3PS        189.22
Aziridine, 1,1',1"-phosphinothioylidynetris-,
Tris(1-aziridinyl)phosphine sulfide     [52-24-4]

» Thiotepa contains not less than 97.0 percent and not more than 102.0 percent of C6H12N3PS, calculated on the anhydrous basis.
Caution — Great care should be taken to prevent inhaling particles of Thiotepa or exposing the skin to it.

Packaging and storage — Preserve in tight, light-resistant containers, and store in a refrigerator.
USP Reference Standards (11) — USP Thiotepa RS.
Identification, Infrared Absorption (197S) — Solution:  3 in 400. Medium: carbon disulfide. Melting range (741): between 52° and 37°
Water, Method I (92I): not more than 2.0%.
Assay
Mobile phase — Prepare a suitable filtered and degassed mixture of water and acetonitrile (9:1). Make adjustments if necessary (see System Suitability under Chromatography (621)).
Standard preparation — Dissolve an accurately weighed quantity of USP Thiotepa RS in Mobile phase to obtain a solution having a known concentration of about 1.5 mg per mL.
Assay preparation — Transfer about 75 mg of Thiotepa, accurately weighed, to a 50-mL volumetric flask, dissolve in Mobile phase, dilute with Mobile phase to volume, and mix.
Resolution solution — Transfer about 10 mg of USP Thiotepa RS to a 4-mL vial, add 2 mL of methanol, and mix. Add 50 µL of 0.1% phosphoric acid solution. Place a cap on the vial, and heat at 65° for 50 seconds. Cool the solution, add 1 mL of methanol, and mix.
Chromatographic system (see Chromatography (621) — The liquid chromatograph is equipped with a 215-nm detector and a 4-mm x 15-cm column that contains packing L1. The flow rate is about 0.8 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed under Procedure: the relative retention times are about 1.25 for methoxythiotepa and 1.0 for thiotepa, and the resolution, R, between the methoxythiotepa peak and the thiotepa peak is not less than 3.0. Chromatograph the Standard preparation, and record the responses as directed under Procedure: the tailing factor for the thiotepa peak is not more than 1.8, the column efficiency is not less than 2600 theoretical plates, and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure — Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C6H12N3PS in the portion of Thiotepa taken by the formula:

50C(ru/rs),

in which C is the concentration, in mg per mL, of USP Thiotepa RS in the Standard preparation, and ru and rs are the thiotepa peak responses obtained from the Assay preparation and the Standard preparation, respectively.”

During the production of Ukrain thiotepa is washed out so that there is no thiotepa in the end product. This has been proved using the most sensitive method of gas chromatography.

 

Stability

The stability of the Ukrain solution for injection was investigated in a real time stability study with 3 batches stored for 60 months at 25°C and 60% relative humidity as well as for 6 months at 40°C and 75% relative humidity (accelerated condition stability study). The study was performed according to the OECD GLP Guidelines. At the end of the study all parameters were within reference range having proved the stability of the end product (Stability Study of Ukrain Ampoules Stored under Controlled Conditions. Final Study Report. March 2003. SGS Lab Simon S.A. Healthcare and Bioscience Services 796.529).