Thiocolchicoside Raw Material Testing Procedure of pharmaceuticals is an integral part of drug development process. From many years, analytical techniques like UV/Vis Spectrophotometry, Spectrofluorimetry, Atomic Absorption Spectrometry, Capillary-Electrophoresis, Liquidchromatography, Gas-chromatography Mass-spectrometry, Luminescence, Voltammetry, Polarography and microbiological testing have been studied for the analysis of pharmaceutical compounds.
1. DESCRIPTION
A yellow crystalline powder.
Place about 10-20 gm sample on clean and dry white butter paper and observe the physical status of material. It should be yellow crystalline powder.
2. SOLUBILITY
Soluble in water and very slightly soluble in ethanol (95%).
2.1 Requirements:
2.1.1 Calibrated Balance.
Procedure
- Weigh accurately a suitable quantity of sample into a glass stoppered test tube and add specified quantity of solvent as per the table given below.
- Shake vigorously to dissolve, keep aside for 15 minutes.
- If sample has not been completely dissolved repeat step no.2.
- Soluble articles, when brought into solution, may show traces of physical impurities, such as minute fragments of filter paper, fibers and other particulate matter, unless limited or excluded by definite tests or other specifications in the individual monograph.
3. IDENTIFICATION
a) By IR
Requirements:
Infrared spectrophotometer, Hydraulic press, Agate mortar·& pestle, Pellet die with holder and Analytical Balance.
Reagent:
Potassium Bromide (Spectroscopy grade)*
Procedure:
- Triturate about l-2mg of the substance to be examined with approximately 300-400mg of dry, finely powdered Potassium Bromide (Spectroscopy grade) in a clean and dry pestle.
Grind the mixture thoroughly, and spread if uniformly in the die and make disc. The disc should be transparent. If the visual inspection shows that the disc is opaque, it should be discarded and remade. - Remove the resultant pellet carefully from the die and mount with the holder in the spectrophotometer.
Record the background spectrum from 4000 to 600 cm-I - Record the spectrum of the sample in the range 4000 :- 600 cm-I.
- Compare it with standard spectrum of reference I working standard.
- The Infrared Spectrum of the sample is concordant with the spectrum of the reference/ working standard.
- If difference appears in the IR spectra of .sample and standard, dissolve equal portion at the test specimen and the reference/ working standard in equal volume of a suitable solvent, evaporate the solution to dryness in similar container under identical conditions and repeat the test on residue.
- Use pre-dried (120°C for 2hrs) potassium bromide for preparation of pallet.
b) By UV
When examined in the range of 220nm to 440nm, a 0.0015%w/v solution in water shows absorption maxima at the same wavelength as obtained with the reference solution.
Requirements:
Ultra Violet Spectrophotometer, Analytical Balance.
Reagents: Purified Water
Procedure:
Preparation of Standard Solution:
Weigh and transfer about 150 mg of Thiocolchicoside !standard into a 100 rhl volumetric flask, dissolve and dilute up to the mark with water. Fu11her dilute 1.0 ml of above solution to 100 ml with water.
Preparation of Sample Solution
Weigh and transfer about 150 mg of Thiocolchicoside sample into I 00 ml volumetric flask, dissolve and dilute up to the mark with water. . Further dilute 1.0ml of above solution to I 00ml with water.
Comparison
Set the wavelength range 220 to 440 nm in the instrument. Rinse the UV cuvette with water and perform baseline correction using water as blank. Fill the cuvette with standard solution and run the scan and record the spectra. Fill the cuvette with sample solution and run the scan and record the spectra. Compare the UV spectra at Maxima and Minima of the sample with standard.
4. pH
(Determined in a 0.5%w/v solution in CO2 free water) Between 6.0 to 7.5
Requirements: pH Meter, beaker, thermometer and Analytical Balance, CO2 free water.
Procedure:
- Calibrate the pH meter as per standard operating procedure for operation and calibration of pH meter as per respective SOP.
- Immerse the electrode in the solution being examined and measure at the temperature 20°C – 25°C (Unless otherwise specified in individual monograph).
- Read the pH value and record.
- Preparation of CO2 free water: Boil adequate quantity of purified water a conical flask at for 15-20 minutes to expel in dissolved carbon dioxide gas. Close the mouth of the flask by keeping the inverted beaker on it to prevent the CO2 laden air going inside the flask while cooling.
5. SPECIFIC OPTICAL ROTATION:
(Determined on 5%w/v solution in water, on dried basis)
Between -550° to -580°
Requirements:
Polarimeter, Analytical Balance.
Reagent: Purified Water
Procedure:
Preparation of Sample Solution:
Transfer about 0.5 g of Thiocolchicoside sample accurately weighed, to· I 00 ml volumetric flask. Add about 80 ml of water, dissolve it completely and make up the volume to 100 ml with the water.
Instrumental Analysis:
Stabilize the Polarimeter for 30 min before taking the reading. Rinse the Polarimeter tube with water and fill the tube with the same make sure that the tube solution is free from air bubbles. Record five reading at 589nm at 23±0.5°C temperature, the scale should show “ZERO” at this point. Rinse the tube with sample solution and fill the tube with same solution. Make sure that the tube solution is free from air bubbles. Record five readings of sample solution at 589nm at 23±0.5°C temperature. Take the average of five readings (a).
Calculations:
Specific Rotation= a x 100
6.RELATED SUBSTANCES (By HPLC, By area percentage normalization)
RELATED SUBSTANCES (By HPLC, By area percentage normalization) Not more than 0.5%
Reagents:
- n-Heptane: HPLC grade
- Chloroform: HPLC grade
- Methanol: HPLC grade
- Glacial acetic acid: AR grade
Chromatographic Conditions:
Instrument A suitable High Performance Liquid Chromatograph (HPLC) equipped with a UV Detector and suitable software for integration.
Column A stainless steel column (25 cm x 4.6 mm), pack with silica (5 µ).
- Detector UV
- Wavelength 360 nm
- Injection volume 20 µI
- Flow rate 0.8 ml/min.
- Run time 60 min.
- Pump mode Gradient
Preparation of solutions:
Test Solution:
Transfer an accurately weighed about 50 mg of Thiocolchicoside sample to a 50ml volumetric flask. Dissolve in methanol and make up to volume and mix.
Reference Solution A:
Transfer an accurately weighed about 50mg of colchicoside standard to a 100 ml volumetric flask. Dissolve in
methanol and make up to volume and mix.
Reference Solution B:
Transfer an accurately weighed about 50mg of Thiocolchicoside standard to a 100 ml volumetric flask. Dissolve in methanol and make up to volume and mix.
Reference Solution C:
Dilute 1.0ml each of reference solution A and reference solution ,B to I 00ml with methanol.
Injection Sequence:
- Inject 20µl of methanol as a blank run and record the chromatogram. No interfering peak should be observed at the retention time of main analyte peak .
- Inject 20µ1 of the reference solution (C) five times and record the chromatograms.
- The theoretical plates for thiocolchicoside peak should not be less than 80000 and tailing feaster for thiocolchicoside peak should not be more than 5.
- The relative standard deviation of areas of the peaks thiocolchicoside in five replicate injections should not be more than 2.0%.
- The relative retention with reference to thiocolchicoside for colchicine is about 0.55, for N-deacetyl N-formyl throcolchicoside is about 1.05 and for colch\coside is about 10.
- Inject 20µ1 of test solution and record the chromatogram.
- In the chromatogram obtained with the test solution the area of each peak corresponding to peak to N-deacetyl N-formyl thiocolchicoside and colchicoside is not more than the area of corresponding
- peak in the chromatogram obtained with reference solution C (0.5 %) and the sum of the area of all other secondary peak is not more than the area of peak due to thiocolchicoside in the chromatogram obtained with reference solution C (0.5 %).
Calculation:
By area percentage normalization.
7.SULPHATED ASH (%w/w, Determined on 1.0g)
Not more than 0.1
Requirements:
Analytical Balance, Muffle furnace, Crucible and desiccator.
Procedure:
Ignite a suitable crucible (Silica, Platinum, and Porcelain) in muffle furnace at temperature 600° ± 50°C for 30 minutes. Take out and keep it in a desiccator, allow it to cool for 15 minutes, weigh it and record the weight of empty crucible. Unless otherwise specified, weigh accurately crucible + sample (about lg) and record the weight. Moisten the sample to be examined with 1 ml of sulphuric acid and heat gently until the sample is completely charred. After cooling moisten the residue with I ml of sulphuric acid, heat gently until white fumes are no longer evolved and ignite at 600±50°C until the residue is completely incinerated. Allow the crucible to cool in desiccator and weigh it and record the weight. Again keep the crucible in muffle for 30 minutes, remove and keep in desiccator, allow it to cool for .15 minutes. Record the weight. If the difference between two successive weighing is more than 0.5 mg, repeat the above procedure until the difference of last two consecutive weight difference is less than 0.5 mg. Record the final weight.
Calculations:
(W2- W)
Sulphated Ash(% w/w) = (W2- W) / (WI – W) X 1OO
Where:
W = Weight of empty crucible
W 1 = Weight of crucible with sample. W2 = Weight of crucible with residue.
8. LOSS ON DRYING (%w/w, Determined on I .0g at I 05°C for 3hrs.)
Not more than 4.0.
Requirements:
Petri dish
Hot air oven thermostatically controlled Desiccator
Electronic weighing balance
Procedure:
Take a preconditioned Petri-dish ( dry at I 05 °C ± 2 ° C for 30 minutes) having capacity about 10 gm . Weigh the Petri-dish. Take accurately about 5-10 gm sample into pre-weighed Petri-dish. Distribute the sample as evenly as practicable. Place the loaded Petri-dish in the drying chamber ( oven) at I 05 °C ± 2 ° C for 10 hours. After drying is completed, open the drying chamber, remove the Petri-dish from it and cool the Petri-dish in desiccators at room temperature. Take the two competitive weighing do not differ by more than two mg, The second weighing being made after an additional two hours of drying under above mentioned condition .
Calculation:
(W1 -W2)
L.O.D. (% w/w) = (W1 -W2) / (W1 -W) x 100
Where
W1= finial mass in gram of weighing bottle with lid along with the properly mixed sample taken for
analysis.
W2= Final mass in gram of weighing bottle & lid with material after drying
W = Mass in gram of empty weighing bottle with lid.
9.ASSAY (By HPLC, %w/w, on dried basis)
Not less than 98.0% and not more than 102.0% of C27H33NO10S.
Reagents:
- Acetonitrile: HPLC Grade
- Water: HPLC Grade (Milli-Q)
- Instrument: A suitable High Performance Liquid Chromatograph (HPLC) equipped with UV Detector and suitable software to integration.
- Column: A stainless steel column (25 cm x 4.6 mm), packed with octylsilane bonded to porous silica (5 µ)
- Detector: UV
- Wavelength: 370 nm
- Injection volume: 20 µI
- Flow rate: 1.0 ml/min. (Linear Gradient)
- Run time: 22 min.
- Pump mode: Gradient
Blank solution:
Use water as blank.
PREPARATION OF SOLUTIONS:
Preparation of Standard Solution:
Accurately weigh and transfer about 100.0mg of Thiocolchicoside working standard in to 100ml volumetric flask. Add about I 0ml water, sonicate to dissolve and make up the volume with water and mix. Further dilute 10ml of the solution to 100ml with water to volume and mix. Filter through 0.45µm or finer porosity membrane filter.
Preparation of sample Solution:
Accurately weigh and transfer about I 00.0mg of Thiocolchicoside sample in to 100ml volumetric flask. Add about I Om! water, sonicate to dissolve and make up the volume with water and mix. Further dilute I 0ml of the solution to I 00ml with water to volume and mix. Filter through 0.45µm or finer porosity membrane filter.
Procedure:
► Inject 20µ1 of diluent as a blank run. Examine the blank -run chromatogram for any extraneous peaks and disregard any corresponding peaks observed in the chromatogram of the sample solution.
► Inject 20µ1 of standard solution in three replicates and calculate the ¾RSD.
► Inject 20µ1 of the sample solution in duplicate into the chromatograph and record the chromatogram.
Evaluation of system suitability:
► The %RSD for three replicate injections of standard solution should not be more than 2.0.
Calculation:
Assay (%w/w, On dried basis)= At Cstd 100 / As Cspl 100-LOD
Where,
At Average area count for Thiocolchicoside peak in the chromatograms of sample solution.
As Average area count for Thiocolchicoside peak in the chromatograms of standard solution.
Cs Concentration of standard.
Cs Concentration of sample.
P Percent potency of Thiocolchicoside working standard, on as is basis.
Precaution:
Equilibrate the column with mobile phase at least 30min before starting the analysis.
10. MICROBIAL LIMIT TEST:
Total viable aerobic count: Not more than I 000 cfu/gram
Fungi (Yeast & Mould) count: Not more than I 00 cfu/gram
Pathogen Test:
E. coli: Should be absent
Solmonella: Should be absent
S. Aureus: Should be absent
P aeruginosa Enterobacteriaceae: Should be absent
* Bacterial Endotoxins Not· more than 87.5 Endotoxin Units per mg of Thiocolchicoside.
PROCEDURE
Suspend 10 gm or 10 ml of the product to be examined in soyabean casein digest medium. A suitable surface active agent such as 0.1 %w/v solution of polysorbate 80 may be added to assist the suspension of poorly wettable substances. ff the product is known to have antimicrobial activity, an inactivating agent may be added to the diluents. If necessary, adjust the pH to about 7.0.and prepare further serial tenfold dilutions using the same diluent.
Total Aerobic Microbial Count:
Dissolve/suspend the specified weight of raw material or specified weight/volume of finished product (for liquid) in 90 ml of soyabean casein digest medium/fluid .. casein soya lecithin pplysorbate-20 (solution A). If necessary homogenize the solution and heat to not more than 40°C with intermittent shaking.
For Total aerobic microbial count aseptically pipette out in duplicate 1.0ml of sample from (solution A) into
sterile Petri plates of90 mm diameter.
Pour 20 to 25 ml of molten soyabean casein digest agar, mix the solution gently.
Allow the plate to solidify and incubate the plates at 30-J5°C for 18 to 24 hours.
After incubation count number of colonies in each plate. Calculate the mean and multiplying with dilution factor.
Negative control:
pour the 20-25 ml of SCDA media into the 90 mm plate without the test sample and incubate along with the test plates.
Positive control:
GPT of the SCDA will be-considering as a positive control and record the result in media preparation record.
Total Yeast and Mould Count:
Dissolve/ suspend the specified weight of raw material or specified weight/volume of finished product (for liquid) in 90 ml of soyabean casein digest medium/fluid casein soya lecithin polysorbate-20 (solution A). If necessary homogenize the solution and heat to not more than 40°C with intermittent shaking.
For total yeast and mould count aseptically pipette in out in duplicate 1.0 ml of sample from (solution A) into
sterile Petri plates of 90mm diameter.
Pour 20 to 25 ml molten sabouraud dextrose agar/ sabouraud chloramphenicol agar, mix the solution gently. Allow the plates to solidify and incubate them at 20-25°C for 5 days.
After incubation count number of colonies in each plate. Calculate the mean and multiplying with dilution
factor.
Negative control: Put the 20-25 ml SDA media into the 90 mm plate in auplicate without the test sample and incubate along with the test plates.
Positive control:
GPT of the SDA will be consider as a positive control and record the result in media preparation record.
Pathogens
Carry out testing for pathogen as follows:
Escherichia coli:
Sample preparation and pre-incubation:
Prepare a sample using a I in IO dilution of not less than l gm/ 1ml of the product to be examined and use 10 ml or the quantity corresponding to I gm or 1 ml to inoculate at 30-35°C for 18-24 hrs.
Selection and subculture:
Shake the container, transfer 1 ml of soyabean casein digest broth to l 00ml of MacConkey broth and incubate at 42-44°C for 24-48 hrs. Subculture on a plate of MacConkey agar at 30-35°C for 18-72 hrs.
Interpretation:
Growth of colonies indicates· the possible presence of E. coli. This is confirming by identification tests. The product complies with the test if no colonies are present or if the identification tests are negative.
Identification test:
Take colony from the MacConkey agar plate and inoculate in 5 ml of peptone tube, incubate at 30-35°C for 18 to 24 hrs. Add 0.5 ml of kovac’s reagent to 0.1 %. Peptone tube for confirmation of indole.
Allow to stand for I minute; if a red colour is produced in the reagent layer, indole is present. It indicates the presence of Escherichia coli.
Positive control: confirm the above test by carrying out test simultane6uly using 10-100 cfu culture suspension for E. coli. The test is invalid if the results do not indicate that the control contains E. coli.
Salmonella:
Sample preparation and pre-incubation:
Prepare the product to be examined and use the quantity corresponding to not less than 10 gm or 10 ml to inoculate a suitable amount of soyabean casein digest broth (solution A), mix and incubate at 30-35°C for 18-24hrs.
Selection and subculture:
Transfer 0.1 ml of soyabean casein digest broth (solution A) to IO ml of Rappaport Vassiliadis Salmonella enrichment broth and incubate at 30-35°C for 24 to 48 hrs. Subculture on plates of Xylose lysine deoxycholate agar. Incubate at 30-35°C for 24 to 48 hrs.
Interpretation:
The possible presence of salmonella is indicated by the growth of well developed, red colonies, with or without black center. This is confirmed by identification tests. The product complies with the test, if colonies of the types described are nor present or if the confirmatory identification tests are negative .
Identification:
If colonies with characteristics described for XLDA is }:)resent, the suspect colonies are transferred to a slant of triple sugar iron agar medium (TSI) using an inoculating wire, by first streaking the surface of the slant, and then stabbing the wire well beneth the s11rface. Incubate at 30-35°C for 24-48 hrs. If the tubes do not have red alkaline slants and yellow acid buts, with or without concomitant blacking of the buts from hydrogen sulfide production, the test specimen meets the requirement for the absence of salmonella .
Positive control: confirm the above test by carry mg out test simultaneously using 10-100 cfu culture suspension of salmonella abony NCTC No. 6017. The test is invalid if the results do not indicate that the control contains salmonella
Staphylococcus aureus:
Sample preparation and pre-incubation:
Prepare a sample using a 1 to 10 dilution of not less than 1 gm of the product to be examined or use 10 ml or the quantity corresponding to I gm or l ml to inoculate a suitable amount of soyabean casein digest broth and mix. Incubate at 30-35°C for 18-24 hrs.
Selection and subculture:
Subculture soya bean casein digest broth (solution A) on a plate of Mannitol salt agar and incubate at 30-35°C for l8-72hrs.
Interpretation:
The possible presence of S. aureus is indicated by the growth of yellow/white colonies surrounded by a yellow zone. This is confirmed by identification tests.
The product complies with the test if colonies of the types described are not present or if the confirmatory identification tests are negative.
Identification:
If characteristic colonies are present, perform coagulase test as follows: Transfer representative colonies to separate tubes containing 0.5ml of rabbit plasma or horse plasma, or any other mammalian plasma. lncubate in a water bath at 37°C. Examine for coagulation after 3 hours of incubation and at suitable intervals up to 24 hours. Comparing with positive and negative controls, the absence of a coagulase reaction indicates the absence of Staphylococcus aureus in the tested article-.
Positive control: Confirm the above test by carrying out test simultaneously using 10-100 cfu culture suspension of Staphylococcus aureus ATC No. 6538. The test is invalid if the results do not indicate that the control contains Staphylococcus aureus.
Pseudomonas aeruginosa:
Sample preparation and pre-incubation:
Prepare a sample using a 1 to 10 dilution of not less than 1 gm of the product to be examined as described in general and use 10 ml or the quantity corresponding to I gm or 1 ml to· inoculate a suitable amount of soyabean casein digest broth and mix. lncubate at 30-35°C for 18-24 hrs.
Selection and subculture:
Subculture soyabean casein digest broth (solution A) on a plate of Cefrimide agar and incubate at 30-35°C for 18-72 hrs.
Interpretation:
Growth of colonies indicates the possible presence of P aeruginosa. This is confirmed by identification tests. The product complies with test, if colonies are not present or if the confirmatory identification tests are negative.
Identification:
Oxidase test: Keep the oxidase disc on cetrimide agar· plates stand for a few minutes, observe the plates if white colour disc converted into purple colour.
This confirmed by identification tests.
Gram staining: Typical colonies of Pseudomonas aeruginosa on gram staining appears as gram negative rods.
Positive control: confirm the above test by carrying out test simultaneously using· 10-100 cfu culture suspension of Pseudomonas aeruginosa ATCCNo.9027. this test is invalid ff the results do not indicate that the control contains Pseudornonas aeruginosa.
Enterobacteriaceae:
Sample preparation and pre-incubation:
Prepare a sample using a 1 in 10 dilution of not less than l gm of the product in soyabean casein digest medium and. incubate at 20-25°C for a time sufficient to results tats, the bacteria but not sufficient to encourage multiplication of the organisms (usually 2 hrs. but not more than 5 hrs.)
Test for absence:
Unless otherwise prescribed, use the volume corresponding to l gm of the product; to inoculate 100ml Enterobacterial Enrichment Broth-Mossel. Incubate at 30-35°C for 24-48 hrs. Subculture on plates of violet red bile glucose agar. incubate at 30-35°C for 18-24 hrs.-The product complies with the test if there is no growth of colonies of Gram Negative Bacteria.
Note: To be carried for injection only.
11 ADDITIONAL TESTS
RESIDUAL SOLVENTS By Gas Chromatography
- Methanol : NMT 3000 ppm
- Ethanol : NMT 5000 ppm
- Chloroform : NMT 60 ppm
- Acetone : NMT 5000 ppm
Chromatography system
- Gas chromatograph: Perkin Elmer GC or equivalent with head space Analyzer,
- Detector : FID
- Column : Capillary, BP 624, 0.53mm x 30m x3urn
- Carrier gas : Nitrogen
- Carrier flow : 1.0 ml per minute
- Operation mode: Split l:10
- Injector Temp : 230°c
- Detector Temp : 250°c
- Column Temp : The column temperature is programmed as follows: 40° hold for 10 minutes, increased at the rate of 20° C/minute to 160°, hold for 4 min
- Injection Volume : 1.0ml
- Head Space Sampler: Vial equilibration temp 100° : Syringe temp 100°
Standard stock solution (a):
In a I 00ml volumetric flask, take about 50ml of dimethylformamide, weigh accurately about 300mg of methanol & 500mg of ethanol, 500rpg Acetone respectively, makeup volume with Dimethylformamide and mix.
Standard stock solution (b):
In a 100 ml volumetric flask, take about 50 ml of dimethylformamide , weigh accurately about 100 mg of chloroform , makeup volume with dimethylformamide and mix.
Standard solution: Take 10ml of standard stock solution (a) & 1 ml of standard stock solution (b) in 100ml volumetric flask & make up volume with dimethylformamide.
Test solution: Take accurately weighs , about 1.0g sample m 10ml volumetric flask, dissolve m dimethylformamide and mix.
Blank Solution: Dimethyl formamide
1. Take 5ml of dimethylformamide in headspace vial, seal the vial with Teflon coated septum &aluminum crimp with the help of crimper.
2. Take 5ml of standard solution in headspace vial, seal the vial with Teflon coated septum and aluminum crimp with the help of crimper (prepare 3 vials).
3. Take 5ml of test solution in headspace vial, seal the vial with Teflon coated septum and aluminum crimp with the help of crimper (Prepare 2 vials).
Procedure:
Separately inject a blank solution, three times standard solution and two times test solutions in the gas Chromatograph and record the peak responses for all the solvents. Calculate the content of residual
Solvents in ppm w/w in the portion ofThiocolchicos1de taken by formula:
Calculation: At x Cs / As x Ct x 100 x 10000
Where:
At =Average area for each sol vents in test solution
As= Average area for each solvents in standard solution
Cs=Concentration for each solvents in standard solution (mg/ml)
Ct=Concentration for test solution (mg/ml)
Note: This test is not valid unless the relative standard deviation of replicate injections of standard Solution for each solvent should not be more than 15.0%
Vital Method : (For Related Substances) Refarmed
Chromatographic System
Column : Water micro porasil (300 mm x 3.9 mm) part no. 27477
Flow Rate : 1.0 ml per min.
Detector : UV 254 nm.
Injection Volume : 20 µI.
Run Time : 30 minutes
Procedure:
Flow the sequence as following for chromatographic impurity
i) One blank injection
ii) Three injection standard solution
iii) Two injection sample solution
Impurity Profile by HPLC
Carry out this analysis by HPLC.
Use Shimadzu LC-20A equipped with Rheodyne injector and Shimadzu SPD-20A UV visible variable wavelength detector. Data acquisition is carried out using Shimadzu class.
Use micro porasil column of300mm length and 3.9 mm ID (part no. 27477 of waters or equivalent).
Effect an isocratic elution with a mixture of chloroform (850 ml), methanol ( 120 ml), formic acid(20 ml) and water (10 ml) as mobile phase. Maintain a flow rate of I ml per minute and carry out the detection at 254 nm .
Sample solution preparation : Transfer about 4.5 to 5.0 mg exactly weighed Thiocolchicoside sample into a IO ml volumetric flask. Dissolve and dilute with mobile phase up to the mark. Filter the solution on a 0.5 micron filter.
Reference Solution Preparation : Transfer about 4.5 to 5.0 mg exactly weighed Thiocolchicoside reference sample into a IO ml volumetric flask. Dissolve and dilute with mobile phase up to the mark. Filter the solution on a 0.5 micron filter.
After conditioning the chromatographic system inject 20d of sample solution and reference solution.
Repeat at last with two more injections. Record the values of the areas corresponding to the peaks of Thiocolchicoside, N-Desacelyl-N-Forrnyl thiocolchicoside and other impurities present in the chromatograms
Obtained with the sample solution. Then calculate the content of each impurify as Thiocolchicoside using the external standard method.