Нестероидни противовъзпалителни средства
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Salicylates (Asrpirin);
Arylalcanoic acids (Indometacin, Diclofenac);
2-Arylpropionic acids (Ibuprofen, Ketoprofen);
N-Arylanthranilic acids (fenamic acids);
Pyrazolidine derivatives (Metamizole);
Oxicams (Piroxicam);
Sulphonanilides (Nimesulide);
COX-2 Inhibitors (Celecoxib);
Други (Omega-3 fatty acids).
NSAIDs
Most NSAIDs act as non-selective inhibitors of the
enzyme
cyclooxygenase,
inhibiting
both
the
cyclooxygenase-1 (COX-1) and cyclooxygenase-2
(COX-2) isoenzymes. Cyclooxygenase catalyses the
formation of prostaglandins and thromboxane from
arachidonic acid (itself derived from the cellular
phospholipid
bilayer
by
phospholipase
A2).
Prostaglandins act (among other things) as messenger
molecules in the process of inflammation. This
mechanism of action was elucidated by John Vane, who
later received a Nobel Prize for this work.
Indometacin
Indometacin contains not less
than 98.5 per cent and not
more than the equivalent of
100.5 per cent of [1-(4chlorobenzoyl)- 5 - methoxy-2methylindol-3-yl] acetic acid,
calculated with reference to
the dried substance.
A white or yellow, crystalline
powder, practically insoluble in
water, sparingly soluble in
alcohol.
Indometacin
IDENTIFICATION
First identification: A, C.
Second identification: A, B, D, E.
A. Melting point (2.2.14): 158°C to 162°C.
B. Dissolve 25 mg in a mixture of 1 volume of 1M hydrochloric acid and 9 volumes of
methanol R and dilute to 100.0 ml with the same mixture of solvents. Dilute 10.0 ml of the
solution to 100.0 ml with a mixture of 1 volume of 1M hydrochloric acid and 9 volumes of
methanol R. Examined between 300 nm and 350 nm (2.2.25), the solution shows an
absorption maximum at 318 nm. The specific absorbance at the maximum is 170 to 190.
C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum
obtained with indometacin CRS. Examine the substances in the solid state without
recrystallisation.
D. Dissolve 0.1 g in 10 ml of alcohol R, heating slightly if
necessary. To 0.1 ml of the solution add 2 ml of a freshly
prepared mixture of 1 volume of a 250 g/l solution of
hydroxylamine hydrochloride R and 3 volumes of dilute
sodium hydroxide solution R. Add 2 ml of dilute
hydrochloric acid R and 1 ml of ferric chloride solution R2
and mix. A violet-pink colour develops.
R R'C = O + H 2 N
R R'C = N
OH
O H. H C l
+ H2O + HCl
Indometacin
Indomethacin
Cl
O
N
CH3
H3CO
COOH
OH-
H
N
CH3
H3CO
+
COO-
O
-O
Cl
Indometacin
E. To 0.5 ml of the solution in alcohol prepared in
identification test D, add 0.5 ml of
dimethylaminobenzaldehyde solution R2. A
precipitate is formed that dissolves on
shaking. Heat on a water- bath. A bluishgreen colour is produced. Continue to heat for
5 min and cool in iced water for 2 min. A
precipitate is formed and the colour changes
to light greyish-green. Add 3 ml of alcohol R.
The solution is clear and violet-pink in colour.
Indometacin
Indomethacin methyl ester
O
R1
N
R1=
C
Cl
CH3
H3CO
COOCH3
1
R2=
N(CH3)2
R2 CHO/H+
H3CO
CH3OH
R2
R1
+
R2
N
H
N
CH3
CH3
H3CO
H3CO
COOCH3
4
- H+
1
6
COOCH3
- H+
Indometacin
1
H 3C
R1
R1
N
CH3
N
COOCH3
H
H3OOC
R2
H3CO
OCH3
7
Fe3+/H+
H3C
R1
R1
N
H3OOC
CH3
N
COOCH3
+
H3CO
R2
5
OCH3
Indometacin
ASSAY
Dissolve 0.300 g in 75 ml of acetone R, through which nitrogen R,
free from carbon dioxide, has been passed for 15 min. Maintain a
constant stream of nitrogen through the solution. Add 0.1 ml of
phenolphthalein solution R. Titrate with 0.1 M sodium hydroxide.
Carry out a blank titration.
1 ml of 0.1 M sodium hydroxide is equivalent to 35.78 mg of
C19H16ClNO4.
STORAGE
Store in a well-closed container, protected from light.
IMPURITIES
A. 4-chlorobenzoic acid.
Sodium diclofenac
Sodium diclofenac contains not less than
99.0 per cent and not more than the
equivalent of 101.0 per cent of sodium 2[(2,6- dichlorophenyl)amino]phenyl]acetate,
calculated with reference to the dried
substance.
A white or slightly yellowish, crystalline
powder, slightly
hygroscopic, sparingly
soluble in water, freely soluble in methanol,
soluble in alcohol, slightly soluble in
acetone, practically insoluble in ether.
Sodium diclofenac
Примеси
Sodium diclofenac
ASSAY
Dissolve 0.250 g in 30 ml of glacial acetic acid R.
Titrate with 0.1M perchloric acid, determining the
end-point potentiometrically (2.2.20).
1 ml of 0.1 M perchloric acid is equivalent to
31.81 mg of C14H10Cl2NNaO2.
STORAGE
Store in a airtight container, protected from light.
Киселинно-основно титруване в ледена оцетна киселина:
неводна среда
Ледената оцетна киселина е един от най-широко използваните разтворители
за определяне на слаби незаредени бази. Тя има силно изразен протондонорен характер, поради което засилва базичния и отслабва киселинния
характер на протолитите. Напр. незаредени бази стават с около 3 рКа
единици по-силни. Ниската диелектрична константа на средата (4.9) води до
силна йонизация и ниска дисоциация на протолитите. Като стандартен
разтвор при титруването на бази се използва перхлорна киселина в оцетна
киселина. При разтварянето протичат следните процеси:
HClO4 + CH3COOH = CH3COOH2+ . ClO4B + CH3COOH = BH+. CH3COOBH+. CH3COO- + CH3COOH2+ . ClO4- = BH+. ClO4- + 2 CH3COOH
Когато базите са във вид на хидрохлориди или хидробромиди се прибавя
разтвор на живачен (ІІ) ацетат. В резултат на тази реакция се освобождават
базите, а хлоридните и бромидните йони се свързват в малко разтворими
съединения.
Ketoprofen
Ketoprofen contains not less than 99.0
per cent and not more than the equivalent
of 100.5 per cent of (RS)-2-(3benzoylphenyl) propionic acid, calculated
with reference to the dried substance.
A white or almost white, crystalline
powder, practically insoluble in water,
freely soluble in acetone, in alcohol and in
methylene chloride.
Ketoprofen
Примеси
Ketoprofen
The chromatographic procedure for Related substances may be carried out
using:
— a stainless steel column 0.15 m long and 4.6 mm in internal diameter
packed with a spherical octadecylsilyl silica gel for chromatography R (5 µm)
with a specific surface of 350 m2·g-1 and a pore size of 0.01 µm (10 nm),
— as mobile phase at a flow rate of 1 ml per minute a mixture of 2 volumes of
phosphate buffer solution pH 3.5 R, freshly prepared, 43 volumes of acetonitrile
R and 55 volumes of water R,
— as detector a spectrophotometer set at 233 nm,
— a loop injector.
Inject 20 µl of reference solution (d). The substances are eluted in the following
order: ketoprofen and ketoprofen impurity A (3-acetylbenzophenone). Adjust the
sensitivity of the detector so that the heights of the two principal peaks in the
chromatogram obtained are not less than 50 per cent of the full scale of the
recorder. The test is not valid unless the resolution between the peaks
corresponding to ketoprofen and ketoprofen impurity A is at least 7.0.
Ibuprofen
Ibuprofen contains not less than
98.5 per cent and not more than
the equivalent of 101.0 per cent of
(RS)-2-(4- isobutylphenyl) propionic
acid, calculated with reference to
the dried substance.
A white, crystalline powder or
colourless
crystals,
practically
insoluble in water, freely soluble in
acetone, in ether, in methanol and
in methylene chloride. It dissolves
in dilute solutions of alkali
hydroxides and carbonates.
Ibuprofen
Ibuprofen
IDENTIFICATION
First identification: A, C.
Second identification: A, B, D.
A. Melting point (2.2.14): 75°C to 78°C.
B. Dissolve 50.0 mg in a 4 g/l solution of sodium hydroxide R and dilute to 100.0 ml
with the same alkaline solution. Examined between 240 nm and 300 nm (2.2.25),
using a spectrophotometer with a band width of 1.0 nm and a scan speed of not more
than 50 nm per minute, the solution shows a shoulder at 258 nm and two absorption
maxima, at 264 nm and 272 nm. The ratio of the absorbance measured at the
maximum at 264 nm to that measured at the shoulder at 258 nm is 1.20 to 1.30. The
ratio of the absorbance measured at the maximum at 272 nm to that measured at the
shoulder at 258 nm is 1.00 to 1.10.
C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the
spectrum obtained with ibuprofen CRS. Examine the substances prepared as discs.
D. Examine by thin-layer chromatography (2.2.27), using silica gel H R as the coating
substance.
Пиразолинови и пиразолидинови производни
ОСНОВНИ СТРУКТУРИ
H
N
H
N
H
N
1
1
N
1
NH
NH
2
2
2
3
3
3
pirazole
3
pirazoline
H
N
1
pirazolidine
O
H
N
1
NH
NH
2
2
3
3
O
O
1,2
dihydro
3H
pyrazol
3
one
pyrazolidine
3,5
dione
Metamizole sodium
A white or almost white,
crystalline powder, very soluble
in water, soluble in alcohol.
Metamizole sodium contains not less than
99.0 and not more than the equivalent of
100.5 per cent of sodium [(1,5-dimethyl-3oxo-2- phenyl-2,3-dihydro-1H -pyrazol-4-yl)N- methylamino]methanesulphonate,
calculated with reference to the dried
substance.
Metamizol sodium
H+
CH3
H3C
N
N
HO3S
N
O
CH3
+
H2O
H2SO3
+H2O
HO3SCH2OH
CH3
H3C
HO
N
CH3
CH3
N
H3C
N
O
HCHO
HN
CH3
N
N
O
CH3
N
H3C
NaO3
S
CH3
H2O
N
N
H2O
O
CH3
H3C
HN
CH3
N
N
+
HCHO + NaHSO3
OH
O2/Cu2+
CH3
CH3
H3C
N
H3C
H2N
O
O2/Cu2+
CH3
HOO
N
N
HN
H3C
N
N
N
O
CHO
O
Metamizole sodium
Impurities
A. R at C4 = NHCHO: 4-formylamino-1,5-dimethyl-2-phenyl-1,2dihydro-3 H-pyrazol-3-one,
B. R at C4 = NH2: 4-amino-1,5-dimethyl-2-phenyl-1,2-dihydro3H - pyrazol-3-one,
C. R at C4 = NHCH3: 4-methylamino-1,5-dimethyl-2-phenyl-1,2dihydro-3 H-pyrazol-3-one,
D. R at C4 = N(CH3)2: 4-dimethylamino-1,5-dimethyl-2-phenyl1,2- dihydro-3 H-pyrazol-3-one.
Metamizole sodium
Related substances Examine by liquid chromatography (2.2.29).
Prepare the solutions immediately before use.
Test solution. Dissolve 50.0 mg of the substance to be examined in methanol R and dilute to
10.0 ml with the same solvent.
Reference solution (a). Dissolve 10.0 mg of metamizole impurity A CRS in methanol R and
dilute to 20.0 ml with the same solvent.
Reference solution (b). Dilute 1.0 ml of reference solution (a) to 20.0 ml with methanol R.
Reference solution (c). Dissolve 40 mg of metamizole sodium CRS in methanol R and dilute
to 20.0 ml with the same solvent.
Reference solution (d). Take 10 ml of reference solution (c) and boil under a reflux
condenser for 10 min. Allow to cool to room temperature and dilute to 20.0 ml with methanol
R.
Reference solution (e). To 6 ml of reference solution (a) add 1 ml of reference solution (c).
The chromatographic procedure may be carried out using:
— a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with
base-deactivated octadecylsilyl silica gel for chromatography R (5 µm),
— as mobile phase at a flow rate of 1.0 ml/min a mixture of 28 volumes of methanol R
and 72 volumes of a buffer solution prepared by adjusting a mixture of 1000 volumes
of a 6.0 g/l solution of sodium dihydrogen phosphate R and 1 volume of triethylamine
R to pH 7.0 with strong sodium hydroxide solution R,
— as detector a spectrophotometer set at 254 nm.
Metamizole sodium
When the chromatograms are recorded in the prescribed conditions, the substances
elute in the following order: impurity A, metamizole, impurity B, impurity C and
impurity D. Inject 10 µl of reference solution (b). Adjust the sensitivity of the system
so that the height of the principal peak in the chromatogram obtained is at least 50
per cent of the full scale of the recorder.
Inject 10 µl of reference solution (d). The chromatogram shows two principal peaks
due to metamizole and impurity C.
Inject 10 µl of reference solution (e). The test is not valid unless in the chromatogram
obtained the resolution between the peaks corresponding to impurity A and
metamizole is at least 2.5.
Inject 10 µl of the test solution and 10 µl of reference solution (b) and continue the
chromatography for 3.5 times the retention time of metamizole. In the chromatogram
obtained with the test solution: the area of any peak corresponding to impurity C is
not greater than the area of the principal peak in the chromatogram obtained with
reference solution (b) (0.5 per cent), the area of any peaks, apart from the principal
peak and the peak due to impurity C is not greater than 0.4 times the area of the
principal peak in the chromatogram obtained with reference solution (b) (0.2 per
cent). The sum of the areas of all the peaks, apart from the principal peak, is not
greater than the area of the principal peak in the chromatogram obtained with
reference solution (b) (0.5 per cent). Disregard any peak with an area less than 0.05
times that of the principal peak in the chromatogram obtained with the reference
solution (b).
Metamizole sodium
Dissolve 0.200 g in 10 ml of 0.01 M hydrochloric
acid previously cooled in iced water and titrate
immediately, dropwise, with 0.05 M
iodine.
Before each addition of 0.05 M iodine dissolve
the precipitate by swirling. At the end of the
titration add 2 ml of starch solution R and titrate
until the blue colour of the solution persists for at
least 2 min. The temperature of the solution
during the titration must not exceed 10°C.
1 ml of 0.05Miodine is equivalent to 16.67 mg of
C13H16N3NaO4S.
Phenylbutazone contains not less
than 99.0 per cent and not more
than the equivalent of 101.0 per
cent of 4-butyl-1,2- diphenylpyrazolidine-3,5-dione, calculated
with reference to the dried
substance.
CHARACTERS
A white or almost white, crystalline
powder, practically insoluble in
water, soluble in ether, sparingly
soluble in alcohol. It dissolves in
alkaline solutions.
Phenylbutazone
A. Melting point (2.2.14): 104°C to 107°C.
B. Dissolve 30.0 mg in 25 ml of methanol R, add 50 ml of 1M sodium
hydroxide and dilute to 100.0 ml with water R. Dilute 5.0 ml of the
solution to 250.0 ml with water R. Examined between 240 nm and 350
nm (2.2.25), the solution shows an absorption maximum at 264 nm. The
specific absorbance at the maximum is 650 to 700. Use as the
compensation liquid a mixture of 0.5 ml of methanol R, 1.0 ml of 1M
sodium hydroxide and 98.5 ml of water R.
C. Examine by infrared absorption spectrophotometry (2.2.24),
comparing with the spectrum obtained with phenylbutazone CRS.
D. To 0.1 g add 1 ml of glacial acetic acid R and 2 ml of hydrochloric
acid R and heat the mixture under a reflux condenser for 30 min. Cool,
add 10 ml of water R and filter. To the filtrate add 3 ml of a 7 g/l solution
of sodium nitrite R. A yellow colour is produced. To 1 ml of the solution
add a solution of 10 mg of b- naphthol R in 5 ml of sodium carbonate
solution R. A reddish- brown to reddish-violet precipitate is forme.
Phenylbutazone
H2O
+ H2O
C6H5
C6H5
C6H5
N NH
H3O+
NH
HOOC
CH
O
COOH
n C4H9
CO2
C6H5
O
n C4H9
t
C6H5
N NH
COOH
+
n C4H9
CO2
n C4H9
HN
C6H5
t
CH2
COOH
Phenylbutazone
Dissolve 0.500 g in 25 ml of acetone R and add
0.5 ml of bromothymol blue solution R1. Titrate
with 0.1M sodium hydroxide until a blue colour is
obtained which persists for 15 s. Carry out a
blank titration.
1 ml of 0.1 M sodium hydroxide is equivalent to
30. 84 mg of C19H20N2O2.
Celecoxib
O
O
S
H2N
N
N
CF3
Pale yellow solid, mp 157
-159 degrees.
H3C
4-[5-(4-Methylphenyl)-3-(trifluoromethyl)-1H -pyrazol-1-yl]benzene sulfonamide
Piroxicam
Piroxicam contains not less than 98.5 per cent and not more than
the equivalent of 101.0 per cent of 4-hydroxy-2-methyl-N-(pyridin2-yl)-2H-1,2-benzothiazine-3-carboxamide 1,1-dioxide, calculated
with reference to the dried substance.
Nimesulide
• Жълтеникав кристален
прах
• неразтворим във вода,
лесно разтворим в
ацетон, трудно
разтворим в етанол
• има полиморфни
форми
• т.т. около149°С
• pKa=6.5
N-(4-Nitro-2-phenoxyphenyl)methanesulphonamide
ASSAY
Dissolve 0.240 g in 30 ml of previously neutralised acetone R and add
20 ml of water R. Titrate with 0.1 M sodium hydroxide, determining the
end-point potentiometrically (2.2.20).
1 ml of 0.1 M sodium hydroxide is equivalent to 30.83 mg of
C13H12N2O5S.
Nimesulide
A: R1 = SO2-CH3, R2 = H, R3 = R4
= NO2 N-(2,4-dinitro-6phenoxyphenyl) methanesulphonamide;
B: R1 = SO2-CH3, R2 = R3 = R4 =
H N-(2-phenoxyphenyl) methanesulphonamide
C: R1 = R2 = R3 = R4 = H
2-phenoxyaniline
D: R1 = R2 = R4 = H, R3 = NO2
4-nitro-2-phenoxyaniline
E: R1 = R2 = SO2-CH3, R3 = R4 =
H N,N-bis(methylsulphonyl)-2phenoxyaniline
F: R1 = R2 = SO2-CH3, R3 = NO2,
R4 = H N,N-bis(methylsulphonyl)-4nitro-2-phenoxyaniline
Nimesulide
•
•
•
•
C18 3.5 µm (150 x 4.6 mm i.d.)
заедно с C18 guard column (10 x 2.1
mm i.d.)
подвижна фаза – ацетонитрил : 50
mM натриево цитратен буфер,
рН=3(53:47)
скорост – 1.1 ml/min
UV-детектор при 240 nm
•
•
•
•
•
•
•
50mmx4.6mm i.d. monolithic column
подвижна фаза – ацетонитрил :
фосфатен буфер, pH=7; 10mM
(34:66, v/v)
•
скорост – 4.0 ml/min
•
•
Hypersil BDS-C18 column
подвижна фаза – 1g/l
динатриевхидрогенфосфат (pH=7.6)
: метанол (56:44)
UV-детектор при 302 nm
Supersphere Select B, 5µM, 125 x
3mm i.d.
подвижна фаза – 1000g метанол,
650g ацетонитрил, 1650g вода, 10g
динатриевхидрогенфосфат, 4g
хептан сулфонова киселина. pH=5.5
(чрез фосфорна киселина 85%)
скорост – 0.7 ml/min
UV-детектор при 297 nm
Omega-3 fatty acids
Omega-3 fatty acids are a family of
polyunsaturated fatty acids which have in
common a carbon-carbon double bond in
the ω-3 position.
Alpha-linolenic acid
Eicosapentaenoic acid
Docosahexaenoic acid
Omega-3 fatty acids
Omega-3 fatty acids
Column 1:
—dimensions: l = 0.3 m, Ø = 7.8 mm;
—stationary phase: styrene-divinylbenzene copolymer R (7 µm), with a
pore size of 10 nm.
Columns 2 and 3 placed closest to the injector:
—dimensions: l = 0.3 m, Ø = 7.8 mm;
—stationary phase: styrene-divinylbenzene copolymer R (7 µm), with a
pore size of 50 nm.
Mobile phase
tetrahydrofuran R.
Flow rate
0.8 ml/min.
Detection
Differential refractometer.
Injection
40 µl.
Styrene (Vinyl benzene) can be copolymerized with other
monomers; for example, divinylbenzene for cross-linking the
polystyrene chains
Divinylbenzene
Omega-3 fatty acids
(Ph. Eur. method 2.2.30)
Size-exclusion chromatography is a chromatographic technique which
separates molecules in solution according to their size. With organic
mobile phases, the technique is known as gel-permeation
chromatography and with aqueous mobile phases, the term gel-filtration
chromatography has been used. The sample is introduced into a column,
which is filled with a gel or a porous particle packing material, and is
carried by the mobile phase through the column. The size separation takes
place by repeated exchange of the solute molecules between the solvent
of the mobile phase and the same solvent in the stagnant liquid phase
(stationary phase) within the pores of the packing material. The pore-size
range of the packing material determines the molecular-size range within
which separation can occur.
Size-exclusion – изключваща по размер
gel-permeation – гел проникваща
gel-filtration – гел филтрираща