VORICONAZOLE METHOD OF ANALYSIS SOP

VORICONAZOLE METHOD OF ANALYSIS SOP

1.0 OBJECTIVE:

To lay down a procedure of analytical report for the active raw material of Voriconazole 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 Characters:

5.1.1 Appearance:

5.1.1.1 White or almost white powder.

5.1.2 Solubility:

5.1.2.1 Material and equipment:

5.1.2.1.1 Glassware (3 test tubes, spatula).

5.1.2.1.2 Acetone.

5.1.2.1.3 Methylene chloride.

5.1.2.1.4 Purified water.

5.1.2.2 Sample:

5.1.2.2.1 Small quantity.

5.1.2.3 Method:

5.1.2.3.1 Take 3 test tubes and add small quantity of sample for testing solubility according to B.P specifications.

5.1.2.3.2 Add purified water in test tube 1, acetone in test tube 2 and methylene chloride in test tube 3 in a small volume and observe the solubility of the sample.

5.1.2.4 Observations:

5.1.2.4.1 The sample in test tube 1 containing with purified water is very slightly soluble.

5.1.2.4.2 The sample in test tube 2 and 3 containing with acetone methylene chloride are freely soluble.

5.2 Assay:

5.2.1 Apparatus:

5.2.1.1 Liquid chromatography apparatus.

5.2.1.2 Glassware (according to the requirement).

5.2.1.3 Spectrophotometer (detector).

5.2.2 Material and reagents:

5.2.2.1 50.0mg of voriconazole CRS.

5.2.2.2 Acetonitrile R.

5.2.2.3 Methanol R.

5.2.2.4 1.90g/L solution of ammonium formate R.

5.2.2.5 Anhydrous formic acid R.

5.2.3 Sample:

5.2.3.1 50.0mg.

5.2.4 Test solution:

5.2.4.1 Test solution (a):

5.2.4.1.1 Take 100ml of beaker and dissolve 50.0mg of the substance to be examined in the mobile phase.

5.2.4.1.2 Sonicate it, if necessary.

5.2.4.1.3 And dilute to 100.0ml with the mobile phase.

5.2.4.1.4 Mix well to ensure complete dissolution.

5.2.4.2 Test solution (b):

5.2.4.2.1 Take 100ml of beaker and add 5.0ml of the test solution (a).

5.2.4.2.2 Dilute it to 100.0ml with the mobile phase.

5.2.5 Reference solutions:

5.2.5.1 Reference solution (a):

5.2.5.1.1 Take 100ml of beaker and dissolve 50.0mg of voriconazole CRS in the mobile phase.

5.2.5.1.2 Sonicate it, if necessary.

5.2.5.1.3 And dilute to 100.0ml with the mobile phase.

5.2.5.1.4 Mix well to ensure complete dissolution.

5.2.5.1.5 Take another 100.0ml of the beaker and add 5.0ml of the above solution in it.

5.2.5.1.6 Dilute it to 100.0ml with the mobile phase.

5.2.6 Column:

5.2.6.1 Size:

5.2.6.1.1 Length=0.15m,

5.2.6.1.2 θ=3.9mm.

5.2.6.2 Stationary phase:

5.2.6.2.1 End-capped octadecylsilyl silica gel for chromatography R (4μm).

5.2.6.3 Temperature:

5.2.6.3.1 35^oC.

5.2.7 Mobile phase:

5.2.7.1 Mix 15 volumes of acetonitrile and 30 volumes of Methanol R and 55 volumes of a 1.90g/L solution of ammonium formate R previously adjusted to pH 4.0 with anhydrous formic acid R while stirring continuously.

5.2.8 Flow rate:

5.2.8.1 1.0ml/min.

5.2.9 Detection:

5.2.9.1 Spectrophotometer at 256nm.

5.2.10 Injection:

5.2.10.1 20μL test solution (b) and reference solution (a).

5.2.11 Run time:

5.2.11.1 3 times the retention time of voriconazole.

5.2.12 Relative retention time:

5.2.12.1 With reference to voriconazole (retention time=about 8 min).

5.2.13 Method of analysis:

5.2.13.1 Firstly prepare the test solution, reference solution and mobile phase according to the requirements.

5.2.13.2 The solutions must be free from solid particles.

5.2.13.3 Prepare the apparatus.

5.2.13.4 The mobile phase solvent mixtures must be deaerated prior to use either by boiling or by applying a partial vacuum to the solvent reservoir.

5.2.13.5 Equilibrate the column with the prescribed mobile phase, flow rate and at temperature specified until a suitable baseline is achieved.

5.2.13.6 Test solution of the mixture to be separated is now introduced into the mobile phase with the help of an injector just before entering the separating column.

5.2.13.7 As the eluate leaves the column it enters a detector, where it is continuously monitored at the specified λ.

5.2.13.8 The electrical signal obtained from detector is amplified and routes to recorder which record the developed chromatogram.

5.2.13.9 Calculate the percentage content of Voriconazole (C₁₆H₁₄F₃N₅O) from the declared content of Voriconazole CRS.

5.2.14 Observations:

5.2.14.1 97.5% to 102.0% (anhydrous substance).

6.0 REVISION LOG:

Revision No.	Effective Date	Reason
00		New SOP

7.0 REFERENCES:

7.1 The British Pharmacopoeia. Vol II., Official Monograph /Voriconazole: 2015, pp. 1170-1172.

8.0 ANNEXURES:

Annexure 1: Observations and calculations of HPLC method.

Annexure: 1

Observations and calculations of HPLC method

Analysis on HPLC

Instrument: ___________________ Date: _________________

Model: ___________________

Column size:	Length=
Column size:	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 (t_m)= _____________

No. of peaks	Retention time of peak of interest (t_r)_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 (t_r)_n	Retention volume = retention time x flow rate

3. Retention factor:

Retention time of unretained peak (t_m)= _____________

No. of peaks	Retention time of peak of interest (t_r)_n	Retention factor of a component k= (t_r-t_m)/t_m

4. Separation factor (α):

No. of peaks	Retention factor of a component (k_n)	Relative retention of two adjacent peaks α = k₂/k₁

5. Resolution:

Retention time of unretained peak (t_m)= _____________

No. of peaks	Retention time of peak of interest (t_r)_n	Width of peak of interest (w)_n	Resolution R_s = 2 (t_r2-t_r1) (w₁-w₂)

6. Efficiency:

No. of peaks or components	Retention time of peak of interest (t_r)_n	Width of peak of interest (w)_n	Efficiency (No. of theoretical plates) N= 16 (t_r/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
No. of peaks or components		At 5%	At 10%	A_s = w_5% 2f	A_s = w_10% 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.,

pic

11. Percentage of content:

Percentage content = (r_U/r_S) x (C_S/C_U) x 100.

r_U= peak response of substance from the sample solution.

r_S= peak response of substance from the standard solution.

C_S= concentration of substance in the standard solution (mg/mL).

C_U= 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
Q.C	Quality control
B.P	British pharmacopoeia
Vol	Volume
QCA	Quality control active ingredient
F	Format
μm	Micron/ micrometer
g/L	Gram per liter
ml	Milliliter
mg	Milligram
CRS	Chemical reference solution
m	Meter
θ	Theta
mm	Millimeter
^oC	Degree Celsius
ml/min	Milliliter per minute
nm	Nanometer
μL	Microliter
Min	Minute
λ	Lamda
%	Percentage

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