CYCLOSERINE USP SOP

CYCLOSERINE USP SOP

1.0  OBJECTIVE:

To lay down a procedure for the active raw material of the Cycloserine USP from the Pharmacopoeial specifications.

2.0  SCOPE:

This SOP shall be applicable in Q.C laboratory.

3.0  RESPONSIBILITY:

3.1  Q.C Analyst.

4.0  ACCOUNTABILITY:

4.1  Q.C Manager.

5.0  PROCEDURE:

5.1  Identification test:

5.1.1        Material and equipment:

5.1.1.1  Glassware (2 test tubes, 1 spatula, 1 beaker).

5.1.1.2  Analytical weighing balance.

5.1.1.3  0.1N sodium hydroxide.

5.1.1.4  3.0ml of 1N acetic acid.

5.1.1.5  Sodium nitroprusside solution.

5.1.1.6  4N sodium hydroxide.

5.1.2        Sample:

5.1.2.1  1.0mg.

5.1.3        Method:

5.1.3.1  Take a test tube add in it about 1.0mg in 10.0ml of 0.1N sodium hydroxide. Dissolve it thoroughly.

5.1.3.2  Take 1.0ml of this resulting solution in another test tube and add 3.0ml of 1N acetic acid and 1.0ml of the mixture, prepared 1 hour before use, of equal parts of sodium nitroprusside solution (1 in 25) and 4N sodium hydroxide.

5.1.3.3  Observe the changes.

5.1.4        Observations:

5.1.4.1  A blue colour gradually develops.

5.2  Specific rotation:

5.2.1        Material and equipment:

5.2.1.1  Polarimeter.

5.2.1.2  Analytical weighing balance.

5.2.1.3  Glassware (1 beaker of 50.0ml, 1 stirrer, 1 spatula).

5.2.1.4  Methanol.

5.2.2        Sample:

5.2.2.1  50mg/ml.

5.2.3        Method:

5.2.3.1  Prepare test solution in a beaker of 100.0ml with 50mg/ml, in 2N sodium hydroxide.

5.2.3.2  Firstly clean the Polarimeter with clean dry cloth,

5.2.3.3  Operate the Polarimeter.

5.2.3.4  Fill the Polarimeter tube with blank solution (2N sodium hydroxide) and determine the observed optical rotation.

5.2.3.5  Similarly, fill the Polarimeter tube with sample test solution and determine the observed optical rotation.

5.2.3.6  Note down the values in annexure-1.

5.2.3.7  Calculate the specific optical rotation by using formula:

[α]_λ ^T = α/lc

5.2.4        Observations:

5.2.4.1  -108^o to -114^o.

5.3  pH:

5.3.1        Material and equipment:

5.3.1.1  Glassware (according to the requirement).

5.3.1.2  pH meter.

5.3.2        Sample:

5.3.2.1  Solution (1 in 10).

5.3.3        Method:

5.3.3.1  Firstly clean the pH meter with clean dry cloth, according to SOP No.

5.3.3.2  Operate the pH meter according to SOP for operation of pH meter i.e.

5.3.3.3  Rejuvenate the electrode before use according to SOP instructions, if there is any need of.

5.3.3.4  Calibrate the electrode of the pH meter according to SOP No.

5.3.3.5  Perform the test on solution (1 in 10).

5.3.3.6  Take a beaker of 100.0ml and add solution (1 in 10) in it. Such that it immersed electrodes in it completely.

5.3.3.7  Maintain the temperature of sample at 25^oC±2^oC.

5.3.3.8  Dip the electrode along with temperature sensor into the sample.

5.3.3.9  When dipping electrode into sample, it must be completely immersed in it.

5.3.3.10    Stir the probe gently in the sample to create a homogeneous sample.

5.3.3.11    Allow the reading to stabilize for a time.

5.3.3.12    Record the observed values of pH & temperature in the respective Annexure-2.

5.3.3.13    Wash the electrodes again after use and store the electrode in storage solution as recommended in SOP of cleaning i.e.. Calibrate the instrument before use.

5.3.4        Observation:

5.3.4.1  Between 5.5 and 6.5.

5.4  Assay:

5.4.1        Apparatus:

5.4.1.1  Glassware (according to requirement).

5.4.1.2  Magnetic stirrer.

5.4.1.3  HPLC apparatus.

5.4.1.4  Analytical weighing balance.

5.4.2        pH 6.8 Phosphate buffer:

5.4.2.1  Prepare pH 6.8 phosphate buffer as given in SOPs of reagents.

5.4.3        Mobile phase:

5.4.3.1  Take a beaker of 1000.0ml and add 0.5g of sodium 1-decanesulfonate in 800 ml of purified water. Dissolve it by using magnetic stirrer operate according to SOP

5.4.3.2  Add 50.0ml of acetonitrile and 5 ml of glacial acetic acid, and mix it again by using magnetic stirrer operate according to SOP

5.4.3.3  Adjust with 1N sodium hydroxide to pH of 4.4.

5.4.3.4  Filter, and degas. Make adjustment if necessary.

5.4.4        Standard preparation:

5.4.4.1  Quantitatively dissolve an accurately weighed quantity of USP Cycloserine RS in pH 6.8 phosphate buffer to obtain a solution having a known concentration of about 0.4mg per ml.

5.4.5        Assay preparation:

5.4.5.1  Take a volumetric flask of 50.0ml and transfer about 20.0mg of Cycloserine, accurately weighed.

5.4.5.2  Dissolve it in sufficient quantity of pH 6.8 phosphate buffer by using magnetic stirrer operate according to SOP

5.4.5.3  Dilute it with the same solution to the volume and mix it by using magnetic stirrer operate according to SOP

5.4.6        Chromatographic system: The liquid chromatography is equipped with:

5.4.6.1  Mode: Liquid chromatography.

5.4.6.2  Detector: 219nm.

5.4.6.3  Column: 4.6-mm × 25-cm column that contains 5-μm packing L1.

5.4.6.4  Column temperature: Maintained at about 30^o

5.4.6.5  Flow rate: 1.0mL/min.

5.4.6.6  Chromatograph the standard preparation, and record the peak responses as directed for procedure

5.4.6.6.1        The tailing factor is not more than 1.8.

5.4.6.6.2        The relative standard deviation for replicate injections is not more than 2.0%.

5.4.7        Procedure:

5.4.7.1  Equilibrate the column and detector with mobile phase at specified flow rate until a constant signal is received.

5.4.7.2  Separately inject equal volumes (about 10μL) of the standard preparation and the Assay preparation into the spectrum.

5.4.7.3  Record the spectrum.

5.4.7.4  Measure the peak responses for Cycloserine.

5.4.7.5  Analyze as directed in the monograph.

5.4.7.6  Calculate the quantity, in μg, of C₃H₆N₂O₂ in each mg of Cycloserine taken by the formula:

50,000 (C/W) (r_U/r_S)

In which,

C = the concentration, in mg per ml, of USP Cycloserine RS in the standard preparation.

W = the quantity, in mg, of Cycloserine taken to prepare the assay preparation.

r_U and r_S = are the peak responses for Cycloserine obtained from the assay preparation and the standard preparation, respectively.

5.4.8        Limit:

5.4.8.1  Cycloserine has a potency of not less than 900μg of C₃H₆N₂O₂.

6.0  REVISION LOG:

Revision No.	Effective Date	Reason
00		New SOP

7.0  REFERENCES:

7.1  USP38NF33 Volume-3 Official Monograph/ Cycloserine: 2015, pp.: 2980.

8.0  ANNEXURES:

Annexure 1: Observations and calculations of specific optical rotation.

Annexure 2: pH measurement.

Annexure 3: Observations and calculations of HPLC method.

Annexure: 1

Observations and calculations of specific optical rotation

Instrument: ___________________                                              Date: _______________

Model: _______________________        Length of Polarimeter tube: ________________

Sample: ________________________________g.

Solvent: ________________________________ml.

Concentration of sample solution: ____________g/ml.

Blank solution:

Sr.#	Blank solution	Temperature	Optical rotation (α)

                                                                                                 Average: _______________

Optical rotation of blank solution: _______________

Sample solution:

Sr.#	Sample solution	Temperature	Optical rotation (α)

                                                                                                 Average: _______________

Optical rotation of sample solution: ______________

Optical rotation of substance = Blank solution - Sample solution.

Specific optical rotation of sample solution by using formula:

[α]_λ ^T = α/lc

                                                                      Result: ________________

Remarks: ___________________________________________________________

Annexure: 2

pH measurement

Sr.#	Temperature	pH

Result: _________________________________________________________________

Annexure: 3

Observations and calculations of HPLC method

Analysis on HPLC Instrument: ___________________                                           Date: _________________ Model: ___________________ Column size: Length= θ= Stationary phase: Temperature: Mobile phase: Flow rate: Injection size: Detector: Wavelength: λ= Sample solution: _______________________ Reference standard solution: ______________ Impurities: ____________________________ (calculate each component calculation separately) OBSERVATIONS: Attach spectrum. CALCULATIONS: 1.      Retention time:                                                                                n= no. of peak Retention time of unretained peak (tm)= _____________ No. of peaks Retention time of peak of interest (tr)n Height of peak of interest (h)n Width of peak of interest (w)n Area of peak of interest A=1/2(h x w) 2.      Retention volume: Flow rate= _______________ml/min. No. of peaks Retention time of peak of interest (tr)n Retention volume = retention time x flow rate 3.      Retention factor: Retention time of unretained peak (tm)= _____________ No. of peaks Retention time of peak of interest (tr)n Retention factor of a component k= (tr-tm)/tm 4.      Separation factor (α): No. of peaks Retention factor of a component (kn) Relative retention of two adjacent peaks α = k2/k1 5.      Resolution: Retention time of unretained peak (tm)= _____________ No. of peaks Retention time of peak of interest (tr)n Width of peak of interest (w)n Resolution Rs = 2 (tr2-tr1)         (w1-w2) 6.      Efficiency: No. of peaks or components Retention time of peak of interest (tr)n Width of peak of interest (w)n Efficiency (No. of theoretical plates) N= 16 (tr/w)2 7.      Height equivalent to a theoretical plate (HETP): Length of column = ________________________ No. of peaks or components No. of theoretical plates (N) Height equivalent to a theoretical plate HETP = L/N 8.      Symmetry factor (tailing factor): No. of peaks or components Distance from the peak max. to leading edge of the peak (f) Width w Symmetry factor At 5% At 10% As = w5%        2f As = w10%        2f 9.      Response factor & Relative response factor: Conc. (mg/ml)= ___________________ No. of peak Peak area Response factor = (peak area/conc.) Relative response factor = (response factor of impurity/response factor of API) 10.  Relative standard deviation (%RSD): Use formula of relative standard deviation where it is required i.e., 11.  Percentage of content: Percentage content = (rU/rS) x (CS/CU) x 100. rU= peak response of substance from the sample solution. rS= peak response of substance from the standard solution. CS= concentration of substance in the standard solution (mg/mL). CU= concentration of substance in the sample solution (mg/mL). RESULTS: ________________________________________________________________________________________________________________________________________________

9.0  ABBREVIATIONS:

Abbreviation	Expanded Form
SOP	Standard operating procedure
&	And
No.	Number
Ltd.	Limited
QCA	Quality control active ingredient
F	Format
Q.C	Quality control
Vol	Volume
g	Grams
ml	Milliliter
Min	Minutes
oC	Degree centigrade
mg	Milligram
M	Molar
%	Percentage
g/L	Gram per liter
N	Normal
USP	United states pharmacopoeia
NF	National formulary
cm	Centimeter
nm	Nanometer
mm	Millimeter
Approx.	Approximately
%	Percentage
μL	Microliter
θ	Theta
λ	Lambda
w/v	Weight by volume
mg/ml	Milligram per milliliter
UV	Ultraviolet
ml/min	Milliliter per minute
o	Degree (angle)
l	Length
g	Grams
c	Concentration (g/ml)
g/ml	Gram per milliliter
α	Alpha
T	Temperature