Theme: Quality control of drugs from group of amino acids of aliphatic series and salts of aliphatic carboxylic acids.
Proteins are essential components of all living matter. As cellular components, proteins perform numerous functions. The chemical reactions fundamental to the life of the cell are catalyzed by proteins called enzymes. Other proteins are structural constituents of protoplasm and cell membranes. Some hormones are characterized as proteins or proteinlike compounds because of their polypeptide structural features.
Proteins are biosynthesized from α-amino acids, and when proteins are hydrolyzed, amino acids are obtained. Some very complex (conjugated) proteins yield other hydrolysis products in addition to amino acids. α-Amino acids are commonly characterized with the generalized structure:
The relative high melting point, solubility, behavior, and acid-base properties characteristic of amino acids can be accounted for on the basis of the dipolar ion structure (commonly called zwitterion). Amino acids in the dry solid state are dipolar ions (inner salts). Amino acids when dissolved in water can exist as dipolar ions.
Amino acids as drugs
Amino Acids – derivatives of carboxylic acids, which contained in structure one or more amino-groups –NH2. They are bifunctional compounds, because consists in moleculas carboxylic groups –СООН (acid groups) and aminogroups –NH2 (basic groups).
Amino Acids are very important biological activity compounds. α– Amino acids are structure parts of proteins and they are more in the nature. Proteins are essential components of all living matter. As cellular components, proteins perform numerous functions. The chemical reactions fundamental to the life of the cell are catalyzed by proteins called enzymes. Other proteins are structural constituents of protoplasm and cell membranes. Some hormones are characterized as proteins or proteinlike compounds because of their polypeptide structural features.
Proteins are biosynthesized from α-amino acids, and when proteins are hydrolyzed, amino acids are obtained. Some very complex (conjugated) proteins yield other hydrolysis products in addition to amino acids.
From proteins hydrolyzaters peoples synthesed more then 20 α-amino acids with the generalized structure:
There are 10 essential (indispensable) amino acids: arginine, valine, histidine, isoleucine, leucine, lysine, methionine, threonine, tryptophan, phenyl alanine. They cannot be synthezed in an organism, and should arrive with food. Proteines as one of three basic compound foodstuff (together with fats and carbohydrates) under the influence of enzymes hydrolyze to separate amino acids, from which in an organism are synthesised specific to given organ proteines.
The greatest quantity of certain amino acids contains in such albumens: 1) glutaqmic acid – gliadic wheat (45,7 %),b-lactoglobulin, miosin (muscles protein), casein (milk protein); 2) cysteine – proteins of integumentary tissue (a horn, a wool, hair, a feather); 3) cystine (for synthesis cysteine) – a horn (6–7 %), human hair (13–14 %); 4) methionine – β-lactoglobulin, miosin, casein.
Types of amino acids
1. Depending on the nature of radical R of amino acid divide on aliphatic, aromatic and heterocyclic.
Aliphatic amino acids contain radical R – opened carbocylic chain, for example:
cysteine
Aromatic – derivatives of benzene, for example:
tyrosine
Heterocyclic – contain in cycles, except Carbon atoms, heteroatoms, for example, imidazole ring contains in molecule of histidine:
histidine
2. Depending on position amino-group–NH2 amino acids divide on: α – β – γ – δ-amino acids, etc. Examples:
α-alanin; β-alanin (is a part some vitamins);
γ-aminobutyric acid, its name as drug is aminalonum (at infringement of brain blood circulation);
ε-aminocaproic acid, apply to a bleeding stop at surgical operations, in particular in obstetrics.
3. Depending on quantity of amino groups–NH2 and carboxylic groups-COOH amino acids divide thus:
monoaminomonocarboxylic acids, which water solutions have the neutral medium (рН 7 7) (рН = 5,5 – 6,5), e.g., methionine:
monoaminodicarboxylic acids, which water solutions have acid reaction (рН 7 7) (рН = 3,1–3,7), e.g., glutamic acid:
diaminomonocarboxylic acids (рН 7), e.g., lysine (it ε-aminocaproic acid which besides in α-position has -NH2-group):
Properties of amino acids
The relative high melting point, solubility, behavior, and acid-base properties characteristic of amino acids can be accounted for on the basis of the dipolar ion structure (commonly called zwitterion). Amino acids in the dry solid state are dipolar ions (inner salts). Amino acids when dissolved in water can exist as dipolar ions.
Amino acids have amphoteric character because of presence in their molecules of acid group-COON and the basic amino group–NH2. Therefore amino acids form salts both at interaction with acids, and with alkalis:
In aromatic amino acids acid properties are expressed more strongly, than the cores that is caused by presence benzene rings.
At the expense of mutual influence of groups-SOON and–NH2 amino acids in water solutions (and even in a firm condition) exist in the form of bipolar ions (tsvitter-ions, internal salts):
(Melting point 220–315 ˚С)
Solubility
Aliphatic amino acids more often soluble in water:
– freely soluble: glycine, alanine, serine, cysteine, acetylcysteine, ornithine, lysine;
– soluble: valine, threonine, methionine;
– sparingly soluble: leucine, isoleucine;
– slightly soluble: aspartic acid, glutamic acid in cold water (freely soluble in boiling water).
They are very slightly soluble or practically insoluble in organic solvents (96 % alcohol, ether).
In solutions of mineral acids and alkalis it is dissolved: aspartic acid, leucine, isoleucine.
Aromatic amino acids are more difficultly dissolved in water:
– very freely soluble: histidine hydrochloride;
– sparingly soluble: phenyl alanine, tryptophan;
– soluble: histidine;
– very slightly soluble: tyrosine.
Very slightly soluble in 96 % spirit and ether: phenyl alanine, tyrosine, histidine, histidine hydrochloride, tryptophan.
Soluble in solutions of mineral acids and alkalis: tyrosine, tryptophan.
Chemical properties
1. At heating amino acid forms various products:
a) α-amino acids forms 2,5–diketopiperazines owing to betweenmolecular condensation:
2,5-diketopiperazin
b) β-amino acids as a result deamination (eliminating of NH3-molecules) change to nonsaturated acids:
c) γ-amino acids forms lactams owing to intramolecular ring formation:
lactam
2. Esterification reaction (for carboxylic group-COOH). At interaction with spirits in the presence of concentrated sulphatic acid H2SO4 are formed esters:
3. Alkylation and acylation reactions (for amino-group–NH2):
rest of acid (acyl-)
4. Polycondensation reaction (formation polypeptides):
peptide-group
The certain sequence rests of amino acids, which paired by peptide-bonds, gives primary structure of proteine.
5. Oxidation reaction (various products are formed):
aliphatic amino acids are oxidised by nitrite acid НNО2 to oxyacid (alcoholic acid):
At more rigid oxidation occurs deamination (eliminating of NH3-molecules), decarboxylation (eliminating of СО2) to aldehyde formation (see reaction with ninhydrin):
Aromatic amino acids at presence of nitrite acids НNО2 (NаNО2 + НСl) are diazotized (diazonium salt is formed), which with phenols (e.g., β–naphthol) in the alkaline medium or with amines in the acid medium azo dyes are formed:
diazonium salt
β-naphthol
azo dye
6. Reduction reaction leads to hydroxyl amine formation:
hydroxyl amine
7. Decarboxylation under the influence of alkali and temperature with primary amine formation:
primary amine
8. Reaction with formaldehyde (formalin); N-methilen derivative, containing azomethin-group (schiff bases) –N = CН2 is formed
schiff bases
or in the form of a bipolar ion:
This reaction is used at quantitative definition α-amino acids by method of formol titration (alkalimetry by Serens method). Formaldehyde blocks NH2-group and compound loses amphoteric character (is released carboxylic-group-COOH), then it is possible it titrate with alkali solution.
Identification of amino acids
1. SPU, A. Polarimetry, measurement of specific optical rotation for all amino acids, except glycine, which is optical inactive compound (does not contain chiralic (unsymmetrical) atom of Carbon.
2. Formation of intracomplex compounds with ions of heavy metals, in particular Co, Аg, Нg, Copper salts:
a) α-amino acids forms very strong chelates with dark blue colour:
dark blue colour
b) β-amino acids with Сu ² + salts also form the painted complexes, but they very not strong;
c) γ-amino acids of this reaction do not give.
3. Reaction with ninhydrin – the general reaction of identification for all amino acids, distinguishing them from peptides and proteines.
With ninhydrin it is possible to present reaction by such two stages.
a) Nihydrin – 1,2,3-triketohydrindenhydrate – at the first stage oxidizes amino acid (destroys of molecule) with formation of ammonia NH3, carbonic gas СО2 and aldehyde, reduces to diketooxyhydrinden.
b) At the second stage the allocated ammonia NH3 is condensed with ninhydrin and with a product of its reduction – diketooxyhydrinden with formation enol form of diketohydrindenketohydrinamine, which in the presence of ammonia NH3 forms ammonium salt of blue-violet colour.
Chemism of reaction amino acids with ninhydrin:
ammonium salt of enol form of diketohydrindenketohydrinamine
with blue-violet colour
4. Specific reactions for sulphurcontaining amino acids (cysteine, acetylcysteine, methionine, cystine):
a) Reaction with sodium nitroprusside Na2 [Fe (CN) 5NO] in the alkaline medium (NаОН, NH3 conc.). In these conditions is formed ionogenic-bonded Sulphur S2 – (see methionine), which reacts with Na-nitroprusside:
S2– + [Fe(CN)5NO] 2– ® [Fe(CN)5NOS] 4–
Cysteine (NаОН medium) gives in this reaction yellow colour, which passing in red; acetylcysteine (NH3 medium) – dark-violet; methionine (NаОН medium, then Н3РО4 + НСl) – dark red colour.
b) Oxidation of Sulphur-ion S2 – to SО42– in a molecule of cysteine under the influence of concentrated solution Н2О2 and FeCl3 with the subsequent revealing sulphate-anions by means of Ва2 +-cations:
SO42 – + Ba2 + ® BaSO4¯
white ppt
ASSAY
1. Keldal method based on definition of the Nitrogene maintenance in organic compounds after its mineralization.
a) Substance of drug mineralized in the special instrument by boiling at presence of potassium sulphate К2SO4, copper sulphate СuSO4 and concentrated sulphuric acid Н2SO4. Thus the amino acid molecule destroys and Nitrogene passes in ammonium hydrogensulphate NH4НSO4.
b) At addition of 30 % sodium hydroxide solution NaOH ammonia NH3 is formed:
NH4HSO4 + 2NaOH ® NH3 + Na2SO4 + 2H2O
b) Received ammonia NH3 drive away in a flask-receiver which contains boric acid solution H3ВО3. Ammonium metaborate NH4ВО2 and ammonium tetraborate (NH4) 2В4О7 are formed.
NH3 + H3BO3 = NH4BO2 + H2O
2NH3 + 4H3BO3 = (NH4) 2B4O7 + 5H2O
d) The formed salts, which рН > 7, it is possible titrate with hydrochloric acid solution НСl (acidimetry, direct titration) in the presence of the mixed indicators (a mix of methyl red and methylene dark blue (2:1)):
NH4BO2 + HCl + H2O = NH4Cl + H3BO3
(NH4) 2B4O7 + 2HCl + 5H2O = 2NH4Cl + 4H3BO3
It is possible to use a method of acidimetry, back titration: to received borates add surplus of a standard solution of hydrochloric acid НСl and not reacted part of НСl titrate with standard solution of sodium hydroxide NаОН at presence phenophthalein (before occurrence of pink colour).
In parallel spend control experience.
The equivalent weight of a substance depends on quantity of Nitrogen atoms in an investigated preparation. As studied amino acids are monoaminocarboxylic acids, therefore them
Еm = M.m.
SPU. Changes in methodical of Nitrogen definition after mineralization by sulphuric acid
According to SPU, ≈ 40 ml distillate (NH3) collect in flask, which contain 20,0 ml
NH3 + HCl = NH4Cl + H2O
Formed distillate (NH4Cl) titrate with
NH4Cl + NaOH = NaCl + NH4OH
HCl + NaOH = NaCl + H2O
Test do again, used in place of test substance 50 mg of glucose R.
Content (%) of Nitrogen calculated by formula:
% N = 0,01401 (n2–n1) / m,
there:
m – mass of shot of test substance, g;
n1 – volume of
n2 – volume of
2. SPU. Acid-base titration. For various amino acids various techniques of acid-base are offered.
1) Direct acidimetry in the aqueous medium (titrant – standard solution of hydrochloride acid НСl) with potentiometric fixing of end point (use for histidine, which water solution has alkaline reaction рН = 7,0–8,5).
Еm = M.m.
2) Direct alkalimetry in the aqueous medium (titrant – standard solution of sodium hydroxide NаОН) with potentiometric fixing of end point (use for histidine hydrochloride, рН = 3,0–5,0); as indicator use bromthymol dark blue (titrate from yellow before dark blue colour) (use for aspartic acid, рН 4,4,0; glutamic acid, рН = 3,1–3,7).
Еm = M.m.
3) Direct acidimetry in the non-aqueous medium (titrant – perchloric acid НСlО4, medium – mix of non-aqueous formic acid НСООН and non-aqueous ice acetic acid СН3СООН in the ratio 1:10) with potentiometric fixing of end point (glycine, phenyl alanine, tyrosine, threonine, methionine, ornithine hydrochloride, lysine hydrochloride); in the presence of the indicator naphthol benzyl (titrate from brownish-yellow before green colour) (alanine, serine, tryptophan, valine, leucine, isoleucine).
Еm = M.m.
3. Alkalimetry by Serenc method (formal titration).
Direct alkalimetry of free monocarboxylic α-amino acids is complicated, as they exist in the form of bipolar ions R–CH(NH3 +)-COO–, they do not contain free carboxylic group-COOH. Therefore to solution of amino acid add formaldehyde solution НСНО (formalin), neutralized by phenolphthalein. It is thus formed N-methylene derivative and carboxylic group-COOH becomes free, which it is possible titrate with standard solution of alkali NaOH in the presence of phenolphthalein as indicator (before occurrence of pink colour).
.
The equivalent weight (Em) of amino acids depends oumber of carboxylic groups -COOH in the investigated drug:
– for monoaminomonocarboxylic acids Еm = m. M.;
– for monoaminodicarboxylic acids (for example, glutamic acid) Еm = M.m. /2.
4. Redox titration for Sulphur-containing amino acids (use for cysteine, acetylcysteine).
1) SPU. Iodometry
a) Iodometry, direct titration (acetylcysteine). Exact shot of drug substance dissolve in water R, add dilute hydrochloric acid НСl, cool in an ice cooler, add potassium iodide solution КI and titrate with standard solution of iodine І2 in the presence of starch solution (before occurrence of dark blue colour).
Еm = M.m.
b) Iodometry, back titration (cysteine, methionine). Exact shot of drug substance dissolve in dilute hydrochloric acid НСl R, cool in an ice cooler, add crystalline potassium iodide КІ and surplus of standard solution of iodine І2. After standing of test solution during 15 mines in the place protected from light, not reacted iodine І2 titrate with standard solution of sodium thiosulphate Na2S2O3, using in the end of titration as indicator starch solution (before disappearance of dark blue colour).
cysteine cystine
I2 + 2Na2S2O3 = 2NaI + Na2S4O6
not reacted
In parallel spend control experience.
2) Iodochlorometry, back titration (methionine). To an investigated solution of test substance add surplus of iodomonochloride standard solution ІCl, as indicator use starch solution. Not reacted iodomonochloride ІCl react with formed iodide acid НI with allocation of iodine І2, which titrate with standard solution of sodium thiosulphate Na2S2O3 before disappearance of dark blue colour.
surplus
ICl + HI = I2 + HCl; I2 + 2Na2S2O3 = 2NaI + Na2S4O6
rest
Еm = M.m.
Application of amino acids
Application of amino acids in medical practice is based on their participation in a nitrogenous exchange, in synthesis necessary for normal functioning of an organism of fibers, пептидов, enzymes, hormones, formation of end-products of a nitrogenous exchange (ammonia, urea) and other vital processes. It „the basic building material” specific fabric fibers and others эндогенных connections.
GLUTAMIC ACID
Acidum glutaminicum
GLUTAMIC ACID
C5H9NO4 M. m. = 147,1 g/mol
Glutamic acid contains not less than 98,5 % and no more than 100,5 % (S)-2-aminopentane-1,5-dicarboxylic acid, in recalculation on a nonvolatile solid (solid).
PROPERTIES
The description. A crystal powder of white colour or colourless crystals.
Solubility. Readily soluble in boiling water R, little soluble in cold water Р, it is practically insoluble in acetic acid R, acetone Р, 96 % alcohol R and ether R.
IDENTIFICATION
The first identification: A, B.
The second identification: A, C, D.
A. Specific optical rotation
The substance should correspond to requirements concerning the specific optical reference, specified in section „Tests for cleanliness”. From +30,5 ° to +32,5 °, in recalculation on a nonvolatile solid (solid). Definitions spend, using solution S.
D. Interaction with a solution of sodium hydroxide
To 2,0 ml of solution S, prepared as it is specified in section „Tests for cleanliness”, add 0,1 ml of a solution of phenolphtalein R, from 3,0 ml to 3,5 ml
E. Reaction with a solution of ninhydrin
20 mg of a substance dissolve at heating in 1 ml water R, add 1 ml solution of ninhydrin and heat up; there is a blue-violet colouring:
F. Interaction with resorcine and sulphatic (sulphate) acid with the next addition of a solution of ammonia
2 mg of a substance mix about 2 mg resorcine R and 5 drops of sulphatic (sulphate) acid R, heat up to occurrence of green-brown colouring. Cool, add 5 ml of water R and 5 ml of ammonia a solution R1; there is a red-violet colouring with green fluorescence:
TESTS FOR CLEANLINESS
Solution S.
The specific optical reference. (SPU, p. 22) From +30,5 ° to +32,5 °, in recalculation on a nonvolatile solid (solid). Definitions spend, using solution S.
QUANTITATIVE DEFINITION
Alkalimetry, direct titration
Nearby
Em = М m.
1 ml
STORAGE
In densely corked container, in the place protected from light.
APPLICATION
For treatment of mental and nervous diseases.
Browse: British Pharmacopoeia 2009 SPU
British Pharmacopoeia Volume I & II
Monographs: Medicinal and Pharmaceutical Substances
Methionine
Methionine
General Notices
(Ph Eur monograph 1027)
Methioninum
Amurex
C5H11NO2S
ээ149.2ээ63-68-3
DEFINITION
Methionine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of (2S)-2-amino-4-(methylsulphanyl)butanoic acid, calculated with reference to the dried substance.
SYNTHESIS
More than 3 % of methionine are a part b–lactoglobuline, miosine, casein. For the first time it was obtained by Muller in 1923.
1. Hydrolysis of albumens with the subsequent высаливанием from hydrolyzaters of hydrolysis products (thirosine, leucyne and methionine) by means of sodium chloride. The received amino acids divide by means of method of chromatography.
SPU. Manufacture. If the substance is received as a result of the process including stages of a fermentation, it should stand article tests “Fermentation products”.
2. The synthetic method based on condensation acetylaminomalonic ester with b–methylthyoethanol. Synthesis consists of such stages:
1) Synthesis of acetylaminomalonic ester by nitrozation of malonic esters with the subsequent reduction and acetylation of obtained product (schemes of reactions see acid глютаминовая).
|
2) Synthesis of b-mehylethanol from ethylenglicol:
HO–CH2–CH2–Cl + NaSCH3 H®O–CH2–CH2–S–CH3 + NaCl
3) Condensation acetylaminomalonic ester with b-methylthyoethanol with the next hydrolysis of formed compound:
Yields of product is 80%.
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, soluble in water, very slightly soluble in alcohol.
IDENTIFICATION
First identificationэA, B.
Second identificationэA, C, D.
эA. (BrPh, SPU). Polarimetry. It complies with the test for specific optical rotation (see Tests).
эB. (BrPh, SPU). Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with methionine CRS. Examine the substances prepared as discs.
эC. (BrPh, SPU). Thin layer chromatography. Examine the chromatograms obtained in the test for ninhydrin-positive substances. The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a).
эD. (BrPh, SPU). Identification of methyl sulphide group by means of sodium nitroprusside after heating with alkali solution
Dissolve
2H3С-S-СН2–CH2–CH(NH2)–COOH + 5NaOH ®
® Н3CSNa + Na2S + CH3OH + 2NH3 + 2HO–CH2–CH2–CH(OH)–COONa
Na2S + Na2[Fe(CN)5NO] ® Na4[Fe(CN)5NOS]
Not pharmacopoeial reactions:
- SP X. To the cooled alloy (fusion cake) add water and acidify by dilute sulphatic acid; there is a smell of hydrogen sulphide H2S and mercaptan CH3SH:
Na2S + CH3SNa + 2H2SO4 = H2S + CH3SH + Na2SO4 + NaHSO4
2. SP X. Reaction with ninhydrin; there is a blue-violet colour (see the general reactions of identification of amino acids, reaction 3).
TESTS
Solution S
Dissolve
Appearance of solution
Solution S is clear (2.2.1) and colourless (2.2.2, Method II).
pH (2.2.3)
The pH of solution S is 5.5 to 6.5.
Specific optical rotation (2.2.7)
Dissolve
optical rotation is + 22.5 to + 24.0, calculated with reference to the dried substance.
Ninhydrin-positive substances
Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R.
Test solution (a)эDissolve
Test solution (b)эDilute 1 ml of test solution (a) to 50 ml with water R.
Reference solution (a)эDissolve 10 mg of methionine CRS in a 10 g/l solution of hydrochloric
acid R and dilute to 50 ml with the same acid solution.
Reference solution (b)эDilute 5 ml of test solution (b) to 20 ml with water R.
Reference solution (c)эDissolve 10 mg of methionine CRS and 10 mg of serine CRS in a 10
g/l solution of hydrochloric acid R and dilute to 25 ml with the same acid solution.
Apply separately to the plate 5 µl of each solution. Develop over a path of
mixture of 20 volumes of glacial acetic acid R, 20 volumes of water R and 60 volumes of
butanol R. Allow the plate to dry in air, spray with ninhydrin solution R and heat at
Chlorides
To 10 ml of solution S add 25 ml of water R, 5 ml of dilute nitric acid R and 10 ml of silver
nitrate solution R2. Allow to stand protected from light for 5 min. Any opalescence in the
solution is not more intense than that in a standard prepared at the same time and in the
same manner using 10 ml of chloride standard solution (5 ppm Cl) R (200 ppm). Examine the
tubes laterally against a black background.
Sulphates (2.4.13)
Dissolve
The solution complies with the limit test for sulphates (300 ppm).
Ammonium (2.4.1)
of ammonium standard solution (100 ppm NH4) R.
Iron (2.4.9)
In a separating funnel, dissolve
quantities, each of 10 ml, of methyl isobutyl ketone R1, shaking for 3 min each time. To the
combined upper layers add 10 ml of water R and shake for 3 min. The lower layer complies
with the limit test for iron (10 ppm).
Heavy metals (2.4.8)
lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32)
Not more than 0.5 per cent, determined on
Sulphated ash (2.4.14)
Not more than 0.1 per cent, determined on
ASSAY
1. (BrPh, SPU). Acidimetry, non-aqueous titration with potentiometric fixing of end point
Dissolve
Titrate with
1 ml of
H3С-S-СН2–CH2–CH(NH2)–COOH + HClO4 ® [H3С-S-СН2–CH2–CH(NH3+)–COOH] ClO4–
Em(C5H11NO2S) = М.m.
2. SP X. Keldal metod – definition of the general content of Nitrogen in substance of drug (see the general methods of quantitative definition of amino acids, method 1).
The content of general Nitrogen in drug should be not less than 9,2 % and no more than 9,5 %.
Еm (C5H11NO2S) = M.m.
3. SP X. Iodometry, back titration in the medium of phosphatic buffer solution (solutions of potassium hydrogenphosphate К2НРО4 and potassium dihydrogenphosphate КН2РО4) and presence of potassium iodide KI
Surplus of iodine I2 titrate with standard solution of sodium thiosulphate Na2S2O3, use starch solution as indicator (add in the end of titration) and titrate until disappearance of dark blue colour:
I2 + 2Na2S2O3 = 2NaI + Na2S4O6
surplus
In parallel spend control experience.
Еm (C5H11NO2S) = M.m.
4. Iodochlorometry, back titration. To an investigated solution of drug substance add surplus of standard solution of iodomonochloride ІCl, as indicator starch solution. Not reacted iodomonochloride ІCl reacts with formed iodide acid НI with allocation of iodine І2, which titrate with standard solution of sodium thiosulphate Na2S2O3 before disappearance of dark blue colour
surplus
ICl + HI = I2 + HCl; I2 + 2Na2S2O3 = 2NaI + Na2S4O6
not reacted
Еm (C5H11NO2S) = M.m. /2
5. Alkalimetry by Serens method (formol titration) (see the general methods of quantitative definition of amino acids).
Еm (C5H11NO2S) = M.m.
Storage. In densely corked containers, in the place protected from light.
Application. Irreplaceable amino acid. For treatment of a dystrophy and liver diseases.
Methionine is necessary for growth and nitrogenous balance in an organism. Plays the important role in a metabolism that is caused by presence in its molecule mobile methyl-group Н3С. Apply to treatment and prevention of toxic defeats of a liver (a cirrhosis, a poisoning with chloroform, Arsene’s preparations, benzene); for treatment of the dystrophy resulting albuminous insufficiency at children and adults after a dysentery and other infectious diseases. Apply орально on 0,5–1,5 gt a preparation 3–4 times a day within 1–30 days.
Release forms: a powder, tablets for 0,25
STORAGE
Store protected from light.
Ph Eur
Action and use
Amino acid.
Ph Eur
Browse: British Pharmacopoeia 2009 SPU, N (Cysteine)
British Pharmacopoeia Volume I & II
Monographs: Medicinal and Pharmaceutical Substances
Cysteine Hydrochloride
Cysteine Hydrochloride
General Notices
(Cysteine Hydrochloride Monohydrate, Ph Eur monograph 0895)
Cysteinum
C3H7NO2S,HCl,H2Oэ
э175.6ээ7048-04-6
DEFINITION
Cysteine hydrochloride monohydrate contains not less than 98.5 per cent and not more than the equivalent of 101.0 per cent of (2R)-2-amino-3-sulfanylpropanoic acid hydrochloride, calculated with reference to the dried substance.
SYNTHESIS
Contains in considerable quantities in fibers of integumentary fabrics (a horn – 6–7 %; a wool, hair – 13–14 %; feathers). Цистеин it can be synthesised in an organism from methionine.
1. Reduction of cystine, which can be received from a horn of animals or a wool
cystine cysteine
2. Reaction serine with phosphor pentasulphide (tionization reaction):
a-amino-b-oxypropionic acid a–аmino-b-mercaptopropionic acid
CHARACTERS
A white or almost white, crystalline powder or colourless crystals, freely soluble in water,
slightly soluble in alcohol.
IDENTIFICATION
First identificationэA, B, E.
Second identificationэA, C, D, E.
эA. It complies with the test for specific optical rotation (see Tests).
эB. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum
obtained with cysteine hydrochloride monohydrate CRS. Examine the substances prepared
as discs.
эC. Examine the chromatograms obtained in the test for ninhydrin-positive substances. The principal spot in the chromatogram obtained with test solution (b) is similar in position, colour, and size to the principal spot in the chromatogram obtained with reference solution (b). (see reaction
эD. (BrPh, SPU). Identification of mercapto-group by means of sodium nitroprusside after heating with alkali solution
Dissolve about 5 mg in 1 ml of dilute sodium hydroxide solution R. Add 1 ml of a 30 g/l
solution of sodium nitroprusside R. An intense violet colour develops which becomes
brownish-red and then orange. Add 1 ml of hydrochloric acid R. The solution becomes
green.
эE. (only BrPh). It gives reaction (a) of chlorides (2.3.1).
Cl- + Ag+ = AgCl↓
white ppt
E. (only SPU). Reaction with hydrogene peroxide and iron(ІІІ) chloride solution with the next revealing sulphate-anions.
Technique.
Oxidation S2– ® SO42–
SO42– + Ba2+ ® BaSO4¯
Not pharmacopoeial reaction:
Reaction with Copper(ІІ) salts; black precipitate is formed.
S2– + Cu2+ ® CuS¯
black precipitate
TESTS
Solution S
Dissolve
Appearance of solution
Dilute 10 ml of solution S to 20 ml with water R. The solution is clear (2.2.1) and not more
intensely coloured than reference solution BY6 (2.2.2, Method II).
Specific optical rotation (2.2.7)
Dissolve
optical rotation is + 5.5 to + 7.0, calculated with reference to the dried substance.
Ninhydrin-positive substances
Examine by thin-layer chromatography (2.2.27), using a TLC silica gel plate R.
Test solution (a)эDissolve
ml with the same solvent. To 5 ml of the solution add 5 ml of a 40 g/l solution of
N–ethylmaleimide R in alcohol R. Allow to stand for 5 min.
Test solution (b)эDilute 1 ml of test solution (a) to 50 ml with water R.
Reference solution (a)эDissolve 20 mg of cysteine hydrochloride monohydrate CRS in water
R and dilute to 10 ml with the same solvent. Add 10 ml of a 40 g/l solution of Nethylmaleimide
R in alcohol R. Allow to stand for 5 min.
Reference solution (b)эDilute 2 ml of reference solution (a) to 10 ml with water R.
Reference solution (c)эDilute 5 ml of test solution (b) to 20 ml with water R.
Reference solution (d)эDissolve 10 mg of tyrosine CRS in 10 ml of reference solution (a) and
dilute to 25 ml with water R.
Apply separately to the plate 5 µl of each test solution and reference solutions (b), (c), and (d)
. Develop over a path of
volumes of water R and 60 volumes of butanol R. Dry the plate at
with ninhydrin solution R and heat at
chromatogram obtained with test solution (a), apart from the principal spot, is not more
intense than the spot in the chromatogram obtained with reference solution (c) (0.5 per cent).
The test is not valid unless the chromatogram obtained with reference solution (d) shows 2
clearly separated principal spots.
Sulphates (2.4.13)
Dilute 10 ml of solution S to 15 ml with distilled water R. The solution complies with the limit
test for sulphates (300 ppm).
Ammonium (2.4.1)
50 mg complies with limit test B for ammonium (200 ppm). Prepare the standard using 0.1 ml
of ammonium standard solution (100 ppm NH4) R.
Iron (2.4.9)
In a separating funnel, dissolve
quantities, each of 10 ml, of methyl isobutyl ketone R1, shaking for 3 min each time. To the
combined organic layers add 10 ml of water R and shake for 3 min. The aqueous layer
complies with the limit test for iron (20 ppm).
Heavy metals (2.4.8)
Dissolve
ml with water R. 12 ml of the solution complies with limit test A for heavy metals (10 ppm).
Prepare the standard using lead standard solution (1 ppm Pb) R.
Loss on drying (2.2.32)
8.0 per cent to 12.0 per cent, determined on
0.7 kPa for 24 h.
Sulphated ash (2.4.14)
Not more than 0.1 per cent, determined on
ASSAY
(BrPh, SPU). Iodometry, back titration
In a ground-glass stoppered flask dissolve
Carry out a blank titration.
1 ml of
cysteine cystine
I2 + 2Na2S2O3 = 2NaІ + Na2S4O6
not reacted
Em(C3H7NO2S) = М. m.
2. Iodometry, direct titration. Shot of substance dissolve in solution HCl, add KI and titrate by standard solution I2 in the presence of starch before occurrence of dark blue colouring.
3. SP Х. Keldal method (see general methods of assay for amino acids, method 1).
General content of Nitrogen in drug mast bee not less then 9,2 % and not more then 9,5 %.
Em(C3H7NO2S) = М. m.
4. Alkalimetry by Serens method (formol titration) (see general methods of assay for amino acids) .Em(C3H7NO2S) = М. м.
STORAGE
Store protected from light.
Ph Eur
Action and use
Amino acid.
Ph Eur
For treatment of initial forms of a cataract.
Apply 5 % water solution at initial forms of a cataract. Is a part of eye drops.
.
Browse: British Pharmacopoeia 2009 SPU
British Pharmacopoeia Volume I & II
Monographs: Medicinal and Pharmaceutical Substances
Acetylcysteine
Acetylcysteine
General Notices
(Ph Eur monograph 0967)
Acetylcysteinum
Mucomyst
ACETYLCYSTEINE
C5H9NO3Sээ
163.2ээ616-91-1
DEFINITION
(2R)-2-(Acetylamino)-3-sulfanylpropanoic acid.
Content
98.0 per cent to 101.0 per cent (dried substance).
SYNTHESIS
It is the N-acetyl derivative of L-cysteine.
Acetilation of cysteine, which obtained by reduction of cystine:
CHARACTERS
Appearance
White or almost white, crystalline powder or colourless crystals.
Solubility
Freely soluble in water and in ethanol (96 per cent), practically insoluble in methylene
chloride.
IDENTIFICATION
First identificationэA, C.
Second identificationэA, B, D, E.
Э
A. (BrPh, SPU). Polarimetry. Specific optical rotation (see Tests).
эB. (BrPh, SPU). Melting point (2.2.14):
эC. (BrPh, SPU). Infrared absorption spectrophotometry (2.2.24).
PreparationэDiscs of potassium bromide R.
Comparisonэacetylcysteine CRS.
эD. (BrPh, SPU). Liquid chromatography. Examine the chromatograms obtained in the test for related substances.
ResultsэThe principal peak in the chromatogram obtained with test solution (b) is similar in retention time and size to the principal peak in the chromatogram obtained with reference solution (b).
эE. (BrPh, SPU). Identification of mercapto-group by means of sodium nitroprusside in the medium of concentrated ammonia solution
To 0.5 ml of solution S (see Tests) add 0.05 ml of a 50 g/l solution of sodium nitroprusside R and 0.05 ml of concentrated ammonia R. A dark violet colour develops.
Na2S + Na2[Fe(CN)5NO] ® Na4[Fe(CN)5NOS]
dark violet colour
Not pharmacopoeial reactions:
1. Reaction with ninhydrin; red-violet colour is formed. (see general reaction of identification of amino acids, reaction 3).
2. Formation of ethylacetate
To test solution add ethanol С2Н5ОН and concentrated sulphatic acid Н2SO4; fruit smell of ethylacetate is formed.
R-NHCOCH3 + HOC2H5 ® R-NH2 + CH3COOC2H5
TESTS
Solution S
Dissolve
Appearance of solution
Solution S is clear (2.2.1) and colourless (2.2.2, Method II).
pH (2.2.3)
2.0 to 2.8.
To 2 ml of solution S add 8 ml of carbon dioxide-free water R and mix.
Specific optical rotation (2.2.7)
+ 21.0 to + 27.0 (dried substance).
In a 25 ml volumetric flask, mix
7.5 ml of a 40 g/l solution of sodium hydroxide R, mix and dissolve. Dilute to 25.0 ml with
phosphate buffer solution pH 7.0 R2.
Related substances
Liquid chromatography (2.2.29). Except where otherwise prescribed, prepare the solutions
immediately before use.
Test solution (a)эSuspend
hydrochloric acid and dilute to 100.0 ml with water R.
Test solution (b)эDilute 5.0 ml of test solution (a) to 100.0 ml with water R. Dilute 5.0 ml of
this solution to 50.0 ml with water R.
Test solution (c)эUse test solution (a) after storage for at least 1 h.
Reference solution (a)эSuspend 4.0 mg of acetylcysteine CRS, 4.0 mg of L-cystine R
(impurity A), 4.0 mg of L-cysteine R (impurity B), 4.0 mg of acetylcysteine impurity C CRS and
4.0 mg of acetylcysteine impurity D CRS in 1 ml of
ml with water R.
Reference solution (b)эSuspend 4.0 mg of acetylcysteine CRS in 1 ml of
Reference solution (b)эSuspend 4.0 mg of acetylcysteine CRS in 1 ml of
acid and dilute to 100.0 ml with water R.
Column:э
э— size: l =
э— stationary phase: octadecylsilyl silica gel for chromatography R (5 µm).
Mobile phaseэStir 3 volumes of acetonitrile R and 97 volumes of water R in a beaker; adjust
to pH 3.0 with phosphoric acid R.
Flow rateэ1.0 ml/min.
DetectionэSpectrophotometer at 220 nm.
Injectionэ20 µl, 3 times; inject
Run timeэ5 times the retention time of acetylcysteine (about 30 min).
Retention timeэImpurity A = about 2.2 min; impurity B = about 2.4 min; 2-methyl-2-thiazoline-
4-carboxylic acid, originating in test solution (c) = about 3.3 min; acetylcysteine = about 6.4
min; impurity C = about 12 min; impurity D = about 14 min.
System suitabilityэReference solution (a):
э— resolution: minimum 1.5 between the peaks due to impurities A and B and minimum 2.0
between the peaks due to impurities C and D.
From the chromatogram obtained with test solution (a), calculate the percentage content of the known impurities (T1) and the unknown impurities (T2) using the following equations:
A1=peak area of individual impurity (impurity A, impurity B, impurity C and impurity D)
in the chromatogram obtained with test solution (a);
A2=peak area of the corresponding individual impurity (impurity A, impurity B, impurity
C and impurity D) in the chromatogram obtained with reference solution (a);
A3=peak area of unknown impurity in the chromatogram obtained with test solution
(a);
A4=peak area of acetylcysteine in the chromatogram obtained with reference solution
(b);
m1=mass of the substance to be examined in test solution (a);
m2=mass of the individual impurity in reference solution (a);
m3=mass of acetylcysteine in reference solution (b).
Limits:э
э— impurities A, B, C, D: for each impurity, maximum 0.5 per cent;
э— any other impurity: for each impurity, maximum 0.5 per cent;
э— total: maximum 0.5 per cent;
э— disregard limit: 0.1 times the area of the principal peak in the chromatogram obtained
with reference solution (b) (0.05 per cent); disregard any peak with a retention time of about
3.3 min due to 2-methyl-2-thiazoline-4-carboxylic acid.
Heavy metals (2.4.8)
Maximum 10 ppm.
ppm Pb) R.
Zinc
Maximum 10.0 ppm.
Atomic absorption spectrometry (2.2.23, Method II).
Test solutionэDissolve
same acid.
Reference solutionsэPrepare the reference solutions using zinc standard solution (5 mg/ml
Zn) R, diluting with
SourceэZinc hollow-cathode lamp.
Wavelengthэ213.8 nm.
Atomisation deviceэAir-acetylene flame.
Use a correction procedure for non-specific absorption.
Loss on drying (2.2.32)
Maximum 1.0 per cent, determined on
Sulphated ash (2.4.14)
Maximum 0.2 per cent, determined on
IMPURITIES
Specified impuritiesэA, B, C, D.
эA. L-cystine,
эB. L-cysteine,
эC. N,N-diacetyl-l-cystine,
эD. N,S-diacetyl-l-cysteine.
Ph Eur
ASSAY
(BrPh, SPU). Iodometry, direct titration
Dissolve
1 ml of
Em(C5H9NO3S) = М.m.
Keldal method – difenition of general content of Nitrogen in drug (see general methods of assay for amino acids, method 1).
Em(C5H9NO3S) = М.m.
Alkalimetry by Serens method (formol titration) (see general methods of assay for amino acids) Em(C5H9NO3S) = М.m.
STORAGE
Protected from light.
Action and use
Sulphydryl donor; antidote to paracetamol poisoning; mucolytic.
Ph Eur
It is used primarily to reduce the viscosity of the abnormally viscid pulmonary secretions in patients with cystic fibrosis of the pancreas or various tracheobronchial and bronchopulmonary diseases.
Acetylcysteine is more active than cysteine. Acetylcysteine is most effective in 10% to 20% solutions. It is used by direct instillation or by aerosol nebulization. It is available as a 20% solution of the sodium salt in 10- and 30ml containers. An opened vial of acetylcysteine must be covered, stored in a refrigerator, and used within 48 houts.
Preparation
Acetylcysteine Injection
Ph Eur
Aminalonum
Acidum gamma-aminobutyricum
Gammalonum
Gamma bosoms
H2N-CH2–CH2–CH2–COOH
C4H9NO2
М.m. = 103,12 g/mol
4-Amino-n-butyric Acid 4-Aminobutanoic acid; gamma amino butyric acid; GABA; C4H9NO2 = 103.1 (2835-81-6)
General reagent grade of commerce containing not less than 97% of C4H9NO2.
The chemical name: 4-amino-n-butyric acid, 4-amonobutanoic acid, gamma amino butyric acid, GABA (names of C4H9NO2 as reagent in the BrPh 2007), 4-aminobutyric acid, g-aminobutyric acid.
In the BrPh C4H9NO2 has name 4-amino-n-butyric acid and use, according to Pharmacopoeia as general reagent. 4-Amino-n-butyric acid, according to Pharmacopoeia, contain not less than 97 % of C4H9NO2 (general reagent).
Obtaining
Alkaline hydrolysis pirolidone-2 with the subsequent neutralization of salt of g-aminobutyric acid by means of acetic acid СН3СООН.
CHARACTERS
Appearance
White crystalline powder or colourless crystals with specific smell of amino acids. Hygroscopic.
Solubility
Freely soluble in water, very slightly soluble in ethanol (96 per cent), practically insoluble in chloroform, acetone. рН water solution of aminalonum 6,5 – 7,5.
IDENTIFICATION
1. Ninhydrin reaction; blue-violet colour is formed. (see general reaction of identification of amino acids, reaction 3).
2. Reaction with alkaline solution of copper sulphate CuSO4; chelate compound of intensively dark blue colour is formed. (see general reaction of identification of amino acids).
3. Fusion with KSCN; allocation of hydrogen sulphide H2S, which revealing by means of lead-acetic paper:
S2 – + Pb2 + ® PbS¯
black ppt
4. Heating with alloxan (mesoxalyl urea) in the medium of dimethylformamide (DMFA); bright-crimson colour is formed.
Assay
1. Acidimetry, non-aqueous titration. Тitrant – perchloric acid HClO4, medium – anhydrous acetic acid СН3СООН, indicator – crystal violet (from violet before blue-green colour).
HOOC–(CH2)3–NH2 + HCl4 = HOOC–(CH2)3–NH3+Cl4–
In parallel spend control experience.
Еm (C4H9NO2) = M. m.
2. Alkalimetry, direct titration of an investigated solution of drug substance with standard solution of sodium hydroxide NaOH in the presence of bromthymol dark blue as indicator (from yellow colour to blue-green).
H2N – (CH2)3COOH + NaOH = H2N – (CH2)3COONa + H2O
Еm (C4H9NO2) = M. m.
3. Keldahl method – difenition of general content of Nitrogen in drug (see general methods of assay for amino acids, method 1).
Ем(C4H9NO2) = М.m.
Storage
In densely corked containers, in the dry cool place protected from light.
Application. Nouromediator
The release form: tablets
Calcium gluconate
The gluconic acid used in the preparation of calcium gluconate can be prepared by electrolytic oxidation of glucose as follows:
Gluconic acid is produced on a commercial scale by the action of a number of fungi bacteria, and molds upon 25% to 40% solutions of glucose. The fermentation is best carried out in the presence of calcium carbonate and oxygen to give almost quantitative yields of gluconic acid. The fermentation is complete in 8 to 18 days.
Calcium gluconate occurs as a white crystalline or granular powder, without odor or test. Its solutions are neutral to litmus paper. One gram of calcium gluconate dissolves slowly in about 30 ml of water and in 5 ml of boiling water. It is insoluble in alcohol and in many organic solvents.
The mineral acids that are stronger than gluconic acid will decompose calcium gluconate. It is incompatible with soluble sulphates, carbonates, bicarbonates, citrates, tartrates, salicylates and benzoates.
Identification. A. Calcium gluconate solution yields the reaction characteristic for calcium salts.
B. Solution reacts with iron chloride ieutral medium to form green complex.
Assay. Dissolve
Each ml of 0.05M EDTA is equivalence to
Calcium gluconate fills the need for a soluble nontoxic well-tolerated form of calcium that can be employed orally, intramuscularly, or intravenously. Calcium therapy is indicated in conditions such as parathyroid deficiency (tetany), general calcium deficiency, and when calcium is the limiting factor in increased clotting time of the blood. It can be used both orally and intravenously.
Potassium acetate CH3COOK
Potassium acetate is a white crystalline powder; it is a slightly hydroscopic with characteristic odor of acetic acid and salty test. It is soluble in water and alcohol.
Identification. A. Yields the reaction characteristic of potassium salts:
B. Yields the reactions characteristic of carboxylic acids: reacts with ethyl alcohol to form ether:
C. Acetate-ion reacts with iron (III) chloride solution to form a red complex:
Assay. Carry out a water-free titration. Dissolve the exact amount of potassium acetate in ice acetic acid and titrate with 0.2N hydrochloric acid use a crystalline-violet as an indicator.
Potassium acetate is indicated for the treatment and prevention of potassium deficiency. It also finds usefulness as a diuretic substance.
Sodium citrate (Sodium hydrocitrate for injection)
It is a white crystalline powder or colorless crystals, odorless with salty test.
Identification. A. Yields the reaction characteristic of sodium salts:
B. Sodium citrate reacts with calcium chloride to form calcium salt that is insoluble in boiling water:
Calcium citrate that is obtained is soluble in hydrochloric acid.
Assay. Carry out a neutralization titration after ion exchange chromatography.
Titrate obtained citrate acid with 0.05M potassium or sodium hydroxide.
4% solution of calcium citrate is used for protection of blood coagulation.
Sodium γ -oxibutyrate HOCH2CH2CH2COONa
It occurs as white or slightly yellow crystalline powder with characteristic odor.
Identification. A. Yields a reaction characteristic of sodium salts.
B. Sodium oxibutyrate reacts with hydrochloric acid to form γ-butyrolactone. Extract synthesized γ-butyrolactone with ether and identify by refractive index (1.4280-1.4360).
Assay.
