In the preceding post, we read about barbiturates, its properties, synthesis of barbiturates, classification, mechanism of action, uses, signs and symptoms and how the samples are packed at scene of crime. In this post, we will look at the Qualitative and Quantitative tests performed in the analysis of barbiturates .
Before we start discussing the various tests used to identify barbiturates, let us first talk about the Extraction and sample preparation of barbiturates for Qualitative and Quantitative Analysis.
For Qualitative Analysis
Both the free acids and the salts are soluble in methanol and this is the solvent of choice for sample preparation for presumptive or qualitative analysis.
Method – Grind or Triturate a small quantity of tablet or capsule contents or bulk drug powder with a small amount of methanol sufficient to obtain a solution containing approximately 1 to 20 mg/ml of barbiturate. The extract may be used directly or filtered and evaporated to dryness under a stream of nitrogen.
For Quantitative Analysis
| Capsules and tablets containing barbiturates in the free acid form | Capsules and tablets containing barbiturates in the salt form |
| Combine the contents of a representative number of capsules or tablets. Transfer an accurately weighed amount to a suitably sized volumetric flask and make up to volume with ethyl acetate. The extract may be used directly or an aliquot removed, filtered and evaporated to dryness under a stream of nitrogen. | Dissolve an accurately weighed amount of the representative sample of barbiturate in 10 ml volume of water in a separating funnel. Add 3N HCl drop-wise to acidify the solution. Extract with several 10ml portions of ethyl acetate. Combine these ethyl acetate extracts and filter through glass wool. Bring the filtered extract to a suitable, known volume with ethyl acetate. |
Let us now see the QUALITATIVE TESTS performed for identifying Barbiturat
COLOUR TESTS
- DILLE-KOPPANYI TEST:
| METHOD | OBSERVATIONS | |
| Method [1] | ||
| 1. | Take an appropriate amount of suspected material in a depression spot plate. Add 3 drops of solution 1 followed by 3 drops of solution 2. | Appearance of purple color indicates the positive test for the presence of barbiturates *** Isopropylamine is responsible for the deprotonation of the barbiturate molecule. The purple colour is caused by complex formation between two barbiturate molecules, two isopropylamine molecules around a tetrahedral cobalt(II) |
| Alternate Method [2] | ||
| 2. | Take an appropriate amount of powdered sample (either from a capsule or pulverized tablet) in a test tube. Add 2 ml of solution 1 and shake well. Now, add 1 ml of solution 2 and again shake well. | Appearance of staple red violet color indicates the presence of barbituric acid or one of its derivatives |
Solution 1: Dissolve 0.1gm of cobaltous acetate tetra hydrate in 100 ml of absolute methanol, and then add 0.2 ml of glacial acetic acid.
Solution 2: Mix 5 ml isopropyl amine with 95 ml of absolute methanol.
- KOPPANYI-ZWIKKER TEST
| Method | OBSERVATION |
| The sample to be tested is dissolved in 1ml of ethanol on a test plate to which one drop of solution 1 is added and agitated | Barbiturates give a blue-violet colour. However, a number of other imide and sulphonamide-type drugs also react. |
Solution 1- Prepare a 1 per cent w/v solution of cobalt nitrate in ethanol, then add 10µl of pyrrolidine.
SALT DETERMINATION
For quantitative purposes, it is necessary to know whether the barbiturate is present as a free acid or in a salt form. We can find out by doing solubility test and determining pH of the suspected material.
| Solubility | Observation |
| Small amounts of the suspected material in each of two test tubes are taken. Several drops of water to the first test tube and several drops of ethyl acetate to the second test tube are added. Observe in which solvent the material dissolves. | Free acids are soluble in organic solvents such as ethyl acetate, but are insoluble in water. The salt forms of the barbiturates are readily soluble in water, but are insoluble in ethyl acetate. |
pH determination
| pH determination | Observation |
| Place a small amount of the suspected barbiturate in a test tube and add 1 ml of water. Determine the pH | A pH greater than 8.0 indicates that the barbiturate is present as the sodium or calcium salt |
THIN LAYER CHROMATOGRAPHY
TLC is a commonly used technique for the separation and identification of various drugs.
| Stationary Phase | Activated silica gel |
| Solvent/Mobile/Developing system: | Ethyl acetate, Methanol and 25% Ammonia[85 : 10 : 5] Chloroform and Acetone [80 : 20] Benzene and acetic acid[90 : 10] Dioxane,benzene and ammonium hydroxide [20 : 75 : 5] Chloroform and Acetone [90:10] |
| Visualization methods | 1. UV light at 254 nm both before and after exposure to ammonia vapour. 2. Mercuric chloride-diphenylcarbazone reagent Spray reagent 3. 0.2% aqueous potassium permanganate solution spray Saturated mercurous nitrate spray |
GAS LIQUID CHROMATOGRAPHY
| PACKED COLUMN TECHNIQUE | |
| Without Derivatization: The use of the packed column technique for the analysis of underivatized barbiturates is not recommended because of their decomposition at high temperature and the presence of mild steel component of the tube. | |
| With Derivatization: | |
| Detector | FID |
| Column | 6 ft. |
| Carrier gas | Nitrogen |
| Flow rate | 45-50 ml/min |
| Derivatizing agent | Trimethylanilinium hydroxide 0.2 M in methanol (Meth Elute) |
| CAPILLARY COLUMN TECHNIQUE | |
| Without derivatization | |
| Detector | FID |
| Column | Fused silica |
| Carrier gas | Nitrogen at 1 ml/min |
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
| REVERSE PHASE | |
| Method-1 | |
| Column | 250mm x 4.6 mm ID |
| Mobile Phase | Acetonitrile : Water [ 30 : 70 ] |
| Flow rate | 0.9 ml/min |
| Detection | UV at 220 nm |
| Method – 2 | |
| Column | 150mm x 4.6 mm ID |
| Packing material | Octadecyl-silica HPLC grade |
| Mobile Phase A : | 0.1 M sodium dihydrogen phosphate buffer , Methanol [ 60 : 40 ] pH 3.5 adjusted with phosphoric acid |
| Mobile Phase B | 0.1 M sodium dihydrogen phosphate buffer, Methanol [ 60 : 40] pH 8.5 adjusted with sodium hydroxide solution |
| Flow rate | 2.0 ml/min. |
| Detection | UV at 216 nm |
SPECTROSCOPIC TECHNIQUES
| UV VIS SPECTROSCOPY | |
| Barbiturate derivatives may be studied by scanning the samples in appropriate organic solvents with the help of UV-Vis spectrophotometry. The value of λmax can be compared with the standard value given in the literature or with using the standard sample of barbiturate derivatives. | |
| MASS SPECTROSCOPY | |
| Barbiturate derivatives may be studied by analyzing the samples with the help of Mass spectrophotometry (GC-MS). The value of principal peaks at m/z can be compared with the standard value given in the literature or with using the standard sample of barbiturate derivatives. | |
| Amylobarbitone | 156, 141, 157, 41, 55, 142, 98, 39 |
| Barbitone | 156, 141, 55, 155, 98, 39, 82, 43. |
| Butobarbitone | 141, 156, 41, 55, 98, 39, 142, 155 |
| Phenobarbitone | 204, 117, 146, 161, 77, 103, 115, 118 |
| quinalbarbitone | 171, 43, 143, 41, 128, 55, 141, 159. |
| IR/FTIR SPECTROSCOPY | |
| Barbiturate derivatives may be studied by scanning the samples with the help of IR Spectroscopy/FTIR. The value of principal peaks at wave numbers can be compared with the standard value given in the literature or with using the standard sample of barbiturate derivatives. | |
| Amylobarbitone | 1725, 1696, 1758, 1317, 1240, 850 (KBr disk) |
| Barbitone | 1680, 1720, 1767, 1320, 1245, 875 (KBr disk) |
| Barbituric acid | 1710, 1734, 1750, 1225, 775, 1190. |
| Pentobarbitone | 1685, 1719, 1744, 1315, 1218, 845. |
| Phenobarbitone | 1712, 1684, 1670, 1770, 1310, 1300. |
| Quinalbarbitone | 1559, 1648, 1690, 1298, 1270, 925. |
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