APPLICATIONS OF NMR-SPECTROSCOPY IN PHARMACEUTICAL ANALYSIS
NMR-spectroscopy has been extensively employed for the identification testing as well as quantita-tive analysis of pharmaceutical substances. These two aspects shall be discussed in the sections that follow :
The versatility and ability of NMR to distinctly differentiate nuclei in various intramolecular environ-ments has placed it as the most reliable and dependable technique for carrying out the identification testing of a host of pure drugs. Hence, any apparent deviations of the spectrum of a sample under investigation vis-a-vis the spectrum of the pure and the authentic pharmaceutical substance usually give rise to an enormous information not only confined to the true identity of the substance but also the probable nature of the impu-rities it possesses.
The survey of literature provides ample evidence of the NMR spectra of a good number of medicinal compounds belonging to various categories, namely : sulphonamides ; barbiturates* ; amphetamines** ; steroids ; antihistamines*** ; penicillins and cephalosporins**** to name a few.
A plethora of pure drugs, their respective combinations and their dosage forms have been assayed by NMR-spectroscopy quantitatively by various researchers and the result(s) thus obtained were duly verified and compared with the standard methods prescribed in various official compendia. A few typical examples of such drugs shall be described briefly here :
A given sample containing a mixture of quinidine (I) and hydroquinidine (II) is dissolved in requisite quantity of deutrochloroform (CDCl3) along with 2, 3, 5-triboromothiophene as the internal standard. The quantitative determination is carried out by comparing the peak area attributed by ethylene of (I) at 5.16 ppm to the internal standard peak at 6.93 ppm. The coefficient of variation was found to be 1%.
The analysis of methsuximide (I) is performed in carbon tetrachloride and of phensuximide (II) in 10% v/v dichloromethane in carbon tetrachloride. In this particular analysis hexamethylcyclotrisiloxane (III) is employed as an internal standard for (I) and (II) ; whereas the frequencies are referenced to usual tetra-methylsilane (TNS).
The simultaneous assay of trimethoprim (I) and sulfamethoxazole (II) present either in tablets or powder may be done effectively by NMR method.
Here, a powdered sample comprising 1 mg of (1), 50 mg of (II), and 30 mg of pure 1,4-dinitrobenzene (III) as internal standard is carefully dissolved by heating in 1 ml of dimethylsulphoxide-d6 and subse-quently centrifuged to eliminate solid residues, if any. For trimethoprim (I) : the assay is solely based on the singlets at 3.40 and 3.55 ppm on account of the aromatic and methoxy protons of (I) respectively. For sulfamethoxazole (II) the singlet at 2.3 ppm is particularly due to the methyl group of (II) ; and the singlet at 8 ppm is due to (III). It is, however, pertinent to mention here that the assay results were fairly in agreement with British Pharmacopoeial method of analysis. Finally, the NMR-spectroscopic method coefficient of vari-ation was found to be only 0.9%.
The assay of meprobamate (1) and mebutamate (II) have been accomplished* by using malonic acid as the internal standard and acetone as the solvent. The results obtained were fairly comparable to the lengthy official procedures.
NMR-assay of meclizine (I) and methaqualone (II), besides a number of other potent hypnotics and their corresponding mixtures have been successfully carried out using an external standardization procedure reported**. It is, however, interesting to observe that additional sources of variability are usually incorpo-rated into an assay employing external standardization, and the same has been duly shown in the results thus obtained i.e., a large coefficient of variation to the extent of 4% achieved.
Natural oils like : olive, peanut, sunflower seed contain mostly the triglycerides, which usually give rise to four characteristic sets of signals in their corresponding PMR-spectra due to the resonance of alkenyl protons, namely :
(i) 4, C-1 glyceride methylene protons,
(ii) 1, C-2 glyceride methylene proton,
(iii) Methylene protons directly linked to a double-bond, and
(iv) Remaining protons on saturated carbon atoms.
Hence, it is possible to measure accurately the integration curve given out by the combined C-1, and C-2 glyceride methylene protons that occurs almost separately at 4. Now, employing these as an internal calibration one may determine conveniently the following two vital informations, such as :
(a) the total number of alkenyl protons, which is a measure of degree of unsaturation, and,
(b) the total number of protons, which is a measure of the average molecular weight.
Thus, the iodine values assayed (calculated) from the alkenyl proton integration*** and the corre-sponding molecular weight match quite favourably with the results obtained by Wijs Method as shown below :
In addition to the above cited typical examples there are a quite a few other drug substances which have been duly assayed by NMR-spectroscopy, thus suggesting the versatility of this technique as an impor-tant analytical tool.