NMR regulatory analysis: enantiomeric purity determination for (R)-(-)-desoxyephedrine and antipode methamphetamine.

Regulatory enantiomeric purity direct determination for (S)-(+)-methamphetamine, the widely abused DEA schedule II controlled substance, and (R)-(-)-desoxyephedrine, over-the-counter nasal inhaler decongestant were developed using 400 MHz 1H NMR spectroscopy. The efficient enantiomeric differentiation was obtained using a diamagnetic chiral solvating agent to dissimilarly perturb the NMR spectra of the enantiomeric solutes. Nonequivalence behavior was studied in terms of all variables that affect population and intrinsic spectra of the fast diastereomeric solvates. Assignment of enantiomer configuration was based on the relative field position of the resolved enantiomeric signals. Optimization of experimental conditions provided significant resolved enantiomeric signals suitable for quantification. Utilizing the relative intensities of the corresponding enantiomeric signals of the N-CH3 assigned to (S)-(+)-methamphetamine and (R)-(-)-desoxyephedrine, the analysis of synthetic enantiomeric mixtures by the proposed methods demonstrated excellent agreements with the known values of the enantiomers present. The mean +/- SD recovery values for the (R)-(-)-enantiomer was 99.9 +/- 0.4% of added antipode (n = 7). The optically pure enantiomer was used to establish the minimum amount detected by the proposed NMR spectroscopic method.

NMR regulatory analysis: determination and characterization of S-adenosyl-L-methionine in dietary supplements.

1H NMR methodology is described for the determination and characterization of the dietary supplement S-adenosyl-L-methionine (SAM), recently introduced to the US market, utilizing a 400 MHz spectrometer without the need of pure reference standards. The developed methodology is able to assess chemical structure, differentiate between biologically-active (S)-diastereomer and biologically-inactive (R)-diastereomer, and determine the ratio of each in the dietry supplement formulation. The determination of the percentage of declared SAM was based on the integrals for the methyl proton of 2-methyl-2-propanol served as an internal standard at delta 1.24 and the methine proton H1' of SAM ribose ring at delta 6.06. The percentage of the active diastereomer was calculated from the relative intensities of the sulfonium methyl singlets corresponding to the major component (S)-diastereomer at delta 2.98 and the minor (R)-counterpart at delta 2.93. The accuracy was established by analyzing synthetic mixtures of the analyte and the internal standard. Excellent agreement was verified between the assay results and the quantities of analyte in the mixture. The mean +/- SD recovery values for SAM and its (R)-diastereomer impurity from a set of 10 synthetic mixtures were 99.6 +/- 0.8% and 22.5 +/- 0.1%, respectively. Using 10 lots, the percentage of SAM ranged from 0 to 110.7% of the declared value and the percentage of the active (S)-diastereomer ranged from 0 to 82.3%.

NMR regulatory analysis: determination and characterization of Chinese-herb aristolochic acids.

1H NMR methodology for the simultaneous determination and characterization of the nephrotoxic components of Aristolochia plants aristolochic acid I (AA-I) and aristolochic acid II (AA-II) was developed utilizing a 400 MHz spectrometer without the need of reference standards. The developed methodology is able to differentiate and assess chemical structures of these toxic injurious compounds. The quantity of each was calculated on the basis of the integrals for the signals of the H-7 and H-8 of the phenanthrene ring of AA-I and AA-II at delta7.38 and delta8.31, respectively, and the vinylic protons of the internal standard maleic acid at delta6.06. The accuracy of the method was established through the analysis of synthetic mixtures containing the internal standard maleic acid, with purified AA-I or combined AA-I and AA-II sodium salts. Excellent agreements were verified between the assay results and the quantities in the mixtures. The mean +/- SD recovery values for purified AA-I and combined AA-I and AA-II from two sets of 10 synthetic mixtures were 99.8 +/- 0.6% and 99.6 +/- 0.8%, respectively. The assay of 4 lots of commercial aristolochic acid by 1H NMR spectroscopy indicated AA-I and AA-II contents in the ranges 45.3-97.1% and 0-15.4%, respectively.

Nonequivalence behavior studies for the direct determination of enantiomeric purity and absolute configuration of timolol by NMR.

Direct determination of both the enantiomeric purity and absolute configuration of timolol was accomplished utilizing 1H NMR (400 MHz) spectroscopy with fast diamagnetic chiral solvating agent to dissimilarly perturb the spectra of enantiomeric solutes. Nonequivalence behavior was studied for all variables that affect populations and intrinsic spectra of the diastereomeric solvates. Optimization of the experimental conditions in terms of probe temperature, substrate concentration and solvating agent to substrate molar equivalents provided resolved enantiomeric signals suitable not only for chiral recognition but also for quantification. Enantiomeric impurity was determined on the basis of relative intensities of the tert-butyl methyl protons resonances; the assignment of enantiomeric configuration was based on the relative field positions of these resonances. The analysis of synthetic mixtures of the enantiomers by the proposed NMR method resulted in assay values which agreed closely with the known quantities of each enantiomer in mixtures tested. The mean +/-SD recovery values for the (R)-(+)-enantiomer was 100.0+/-1.6% of added antipode (n = 8). The optically pure enantiomers were used to establish the minimum detection limits of0.1%. The developed methodology represents a rapid and powerful tool for regulatory analysis.

A stability-indicating proton nuclear magnetic resonance spectroscopic method for the analysis of propantheline bromide in pharmaceutical samples.

A rapid, specific and accurate proton nuclear magnetic resonance (1H NMR) spectroscopic method was developed for the simultaneous quantitative analysis of propantheline bromide and its degradation product, xanthanoic acid, in bulk materials and tablets. 1,3,5-Trinitrobenzene served as an internal standard and deuterochloroform was used as the solvent for the analytical samples. The quantities of propantheline bromide and xanthanoic acid were calculated on the basis of the integrals for signals of the methine proton of propantheline at 5.09 ppm, the methine proton of xanthanoic acid at 4.99 ppm, and the aromatic protons of the internal standard at 9.39 ppm. The accuracy of the method was established through the analysis of synthetic mixtures containing the parent compound, its degradation product and the internal standard. An excellent agreement was verified between the assay results and the quantities of the various compounds in the mixtures. The mean +/- SD recovery values for propantheline bromide and xanthanoic acid from a set of 10 synthetic mixtures were 99.6 +/- 0.8% and 98.9 +/- 1.8%, respectively. The assay of 10 lots of commercial propantheline bromide tablets by 1H NMR spectroscopy indicated drug and degradate contents in the ranges 97.1-99.8% and 0.1-0.9%, respectively. In addition, the proposed analytical method was found suitable for detecting the formation of xanthanoic acid from propantheline bromide in aqueous media in concentrations below 0.1% of that of the parent compound.

Direct enantiomeric purity determination of the chiral anesthetic drug bupivacaine by 1H NMR spectroscopy.

The direct determination of the enantiomeric purity of the chiral anaesthetic drug bupivacaine has been developed using 1H NMR (400 MHz) spectroscopy with a chiral solvating agent. Optimization of experimental conditions in terms of temperature, substrate concentration and solvating agent to substrate molar ratio provided two significant signal splittings for chiral recognition resulting from diastereomeric solvation. Based on the relative intensities of the aliphatic methyl resonances assigned to (S)-(-)- and ($)-(+)-bupivacaine, the analysis of synthetic mixtures of the enantiomers by the proposed NMR method resulted in assay values which agreed closely with the known quantities of each enantiomer in the mixtures tested. The mean +/- SD recovery values for the (R)-(+)-enantiomer was 100.0 +/- 0.6% of added antipode (n = 7). The optically pure enantiomers were used to establish the minimum sensitivity of the NMR spectroscopic method of chiral analysis.

A simple method for the identification and assay of iopamidol and iothalamate meglumine in pharmaceutical samples based on proton nuclear magnetic resonance spectroscopy.

A proton nuclear magnetic resonance (PMR) spectroscopic method is described for the direct assay and identification of the triiodinated radiographic contrast agents iopamidol (nonionic type) and iothalamate meglumine (ionic type) in commercial solutions and as a bulk material. Samples were prepared by simply diluting an injectable solution with or dissolving a powdered sample in D2O. Sodium acetate was added to serve as an internal standard. Quantitations were based on the resonance signals for the protons of the CH3-CO-group at 1.58 ppm (iopamidol) or 2.25 ppm (iothalamate), CH3-N-group at 2.38 ppm (meglumine) and CH3-CO-group at 1.92 ppm (acetate). The mean +/- SD (n = sets of 10 samples each) recovery of iopamidol, iothalamic acid and meglumine from synthetic mixtures with the internal standard were 99.6 +/- 0.63, 99.7 +/- 0.66 and 99.9 +/- 1.18%, respectively; with the values ranging from 98.7-100.9% for iopamidol and iothalamate, and from 98.3-100.8% for meglumine.

1H-NMR studies of drugs with chiral solvating agent: the direct enantiomeric purity determination of the chiral anesthetic, prilocaine.

Chiral recognition of prilocaine was obtained on a 400 MHz 1H-NMR spectrometer by fast diastereomeric interactions with the chiral solvating agent (S)-(+)-2,2,2-trifluoro-1-(9-anthryl) ethanol (TFAE). Assignment of absolute configuration was based on relative field position of the resolved enantiomeric signals. Influence of temperature, substrate concentration and solvating agent to substrate molar ratio on enantiomeric resolution were studied and evaluated. Optimization of experimental conditions provided two significant resolved signals for quantitative use. Utilizing the relative intensities of the resolved enantiomeric signals of the methine proton attached to the stereogenic center assigned to (S)-(+)- and (R)-(-)-prilocaine, the analysis of synthetic mixtures of the enantiomers by the proposed NMR method resulted in assay values which agreed closely with the known quantities of each enantiomer in the tested mixtures. The mean +/- SD recovery values for the (S)-(+)-enantiomer was 99.9 +/- 0.2% of added antipode (n = 7). The optically pure enantiomers were used to establish the minimum amount detected by the proposed NMR spectroscopic method.

Optimization of enantiomeric separation for quantitative determination of the chiral drug propranolol by 1H-NMR spectroscopy utilizing a chiral solvating agent.

High-field 1H-NMR methodology for enantiomeric composition determination of the chiral drug propranolol utilizing a chiral solvating agent is reported. Optimal experimental conditions for the resolution of enantiomers were determined by studying the interaction of substrate concentration, chiral solvating agent concentration and temperature. The success of the method is based on the selection of a chiral solvating agent that has the following two characteristics. First, it possesses functional groups that are complimentary to those of the chiral substrate for significant interaction to occur. Second, it has a group of diamagnetic anisotropy near its stereogenic center for translating spatial environments of solute nuclei into different magnetic environments that are measurable by NMR spectroscopy. Optical purities were determined on the basis of the intensities of the methyl proton resonances. The analysis of synthetic enantiomeric mixtures of propranolol by the proposed NMR method resulted in assay values, which agreed closely with the known quantities of each enantiomer in the mixtures tested. The mean +/- SD recovery values for the (R)-(+)-enantiomer was 100.0+/-0.6% of added antipode (n = 7).

Isolation and identification of process impurities in trimethoprim drug substance by high-performance liquid chromatography, atmospheric pressure chemical ionization liquid chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy.

Twenty-two lots of recently synthesized trimethoprim drug substance, from five different manufacturers, in three different countries of origin, China, Israel and the United States, were investigated for the presence of impurities. A liquid chromatographic system, using gradient elution, and a mobile phase consisting of 0.25% TEA/0.1% formic acid (pH 5.8)--acetonitrile, was used to separate and detect two significant, recurring impurities in trimethoprim drug substance. The two impurities were isolated by preparative liquid chromatography and identified, using a combination of liquid chromatography/mass spectroscopy and nuclear magnetic resonance, as 2,4-diamino-5-(4-ethoxy-3,5-dimethoxybenzyl) pyrimidine and 2,4-diamino-5-(3-bromo-4,5-dimethoxybenzyl) pyrimidine. These impurities were not detected by the compendial method and were present at significant levels in 17 of the lots tested. Total impurity concentrations were in the range of 0.1-2.1%.

Determination of enantiomeric composition of ibuprofen in bulk drug by proton nuclear magnetic resonance spectroscopy with a chiral lanthanide chelate.

The enantiomeric composition of ibuprofen was determined in a simple and reliable manner by proton nuclear magnetic resonance spectroscopy with a chiral lanthanide chelate. Optimum complexation with the europium (III) chelate took place in CCl4 after conversion of the enantiomeric sample into a mixture of methyl esters. The optimization of the experimental conditions in terms of substrate concentration and lanthanide chelate to substrate molar ratio led to two sets of signals of utility for quantitative purposes. Analysis of synthetic enantiomeric mixtures by the proposed method demonstrated excellent agreement between the assay results and the known masses of each enantiomer present in the mixture samples. The average +/- S.D. recovery values were 99.39 +/- 0.92 and 99.42 +/- 0.68% (n = 10) of (S)-(+)-ibuprofen depending on whether the quantitation was based on the alpha-methyl protons or ester methyl protons, respectively.

Simple 1H NMR spectroscopic method for assay of salts of the contrast agent diatrizoate in commercial solutions.

A simple, accurate, and specific 1H NMR spectroscopic method was developed for the assay of diatrizoate meglumine or the combination diatrizoate meglumine and diatrizoate sodium in commercial solutions for injection. A mixture of injectable solution and sodium acetate, the internal standard, was diluted with D2O and the 1H NMR spectrum of the solution was obtained. Two approaches were used to calculate the drug content, based on the integral values for the -N-CO-CH3 protons of diatrizoic acid at 2.23 ppm, and -N-CH3 protons of meglumine at 2.73 ppm, and the CH3-CO-protons of sodium acetate at 1.9 ppm. Recoveries (mean +/- standard deviation) of diatrizoic acid and meglumine from 10 synthetic mixtures of various amounts of these compounds with a fixed amount of internal standard were 100.3 +/- 0.55% and 100.1 +/- 0.98%, respectively. In addition to providing a direct means of simultaneously assaying diatrizoic acid and meglumine, the proposed NMR method can also be used to identify diatrizoate meglumine and each of its molecular components.

Determination of the optical purity of timolol maleate by proton nuclear magnetic resonance spectroscopy with a chiral Pr(III) shift reagent.

A 1H NMR spectroscopic method with chiral shift chelate is presented for the determination of the optical purity of timolol maleate. Optimum experimental conditions were established by studying the interactions of solvents (CCl4, CDCl3), substrate concentration, and the type and concentration of chiral lanthanide chelate (Pr(hfc)3, Eu(hfc)3). Larger induced shift (delta delta) and enantiomeric shift difference (delta delta delta) values, and more detailed spectral differences were obtained with Pr(hfc)3 than with Eu(hfc)3. By monitoring the spectral changes of the resonance signals for the enantiomeric -C(CH3)3 protons, suitable conditions for quantitative determinations were found when 0.1 molar equivalents of Pr(hfc)3 were complexed with 0.074 M of substrate. Enantiomeric compositions were calculated from the relative intensities of the enantiomeric -C(CH3)3 proton resonances. Based on the analysis of six synthetic enantiomeric mixtures, the mean +/- SD recovery of (R)-(+)-timolol by the proposed method was 99.5 +/- 1.17% of the amount added.

Determination of ephedrine, pseudoephedrine, and norephedrine in mixtures (bulk and dosage forms) by proton nuclear magnetic resonance spectroscopy.

A simple, specific, and accurate 1H nuclear magnetic resonance (NMR) spectroscopic method has been developed for quantitative determination of the Ephedra alkaloids (-)-ephedrine, (+)-pseudoephedrine, and (+/-)-norephedrine, either singly or in mixtures with each other. Determination of individual alkaloids was carried out in D2O solution, with acetamide as internal standard. Although calculations were based on integrals for the C-CH3 protons, those for the N-CH3 and -CH-O- protons may also be useful, depending on the compound. Determination of diastereomeric cross-contamination of ephedrine and pseudoephedrine--or of the concentrations of these alkaloids in the presence or absence of (+/-)-norephedrine--was feasible by using the integrals for the -CH-O- protons after addition of a trace of DCl. Mean recoveries for ephedrine and pseudoephedrine from their respective synthetic mixtures with the internal standard (acet- amide) were > or = 99.9 +/- 0.6% (n = 10) and 99.6 +/- 0.8% (n = 10) of the amount added. Recovery for pseudoephedrine from diastereomeric mixtures with ephedrine was > 99.4 +/- 0.7% (n = 10) of the amount added, with as little as 1.92% still being measurable. Mean recovery of (+/-)-norephedrine from mixtures with ephedrine and pseudoephedrine was > 99.7 +/- 2.5% (n = 4) of the amount added, with about 1% still being measurable. Application of the proposed NMR spectroscopic method to commercial dosage forms, including ephedrine sulfate injections and pseudoephedrine hydrochloride tablets, yielded assay results ranging from 97.8 to 100.2% (mean, 99.2%) and from 98.7 to 100.5% (mean, 99.7%) of declared, respectively.

Proton nuclear magnetic resonance spectroscopic detection and determination of ethylene glycol dimethacrylate as a contaminant of methyl methacrylate raw material.

A simple, specific, and accurate proton nuclear magnetic resonance (1H NMR) spectroscopic method is presented for detection and assay of ethylene glycol dimethacrylate dimer as a contaminant of methyl methacrylate monomer. In addition to minimizing exposure of the analyst to the irritant and toxic methacrylic acid esters, the proposed method requires no sample preparation. Quantitations are based on integrals for signals of methylene protons of ethylene glycol dimethacrylate at 4.37 ppm and methyl protons of methyl methacrylate at 3.70 ppm. Analysis of 10 synthetic mixtures of the monomer with 1-11% of dimer yielded a dimer recovery of 100.5 +/- 2.05% (mean +/- standard deviation). Correspondence (correlation coefficient, r = 0.9999) between the amount of dimer added and the amount found was excellent. The proposed method measures as little as 1% of dimer.

Determination of the optical purity and absolute configuration of threo-methylphenidate by proton nuclear magnetic resonance spectroscopy with chiral solvating agent.

The direct determination of both the optical purity and absolute configuration of threo-methylphenidate has been accomplished in a simple, specific, and accurate manner by 1H-NMR spectroscopy. The enantiomeric resonances of threo-methylphenidate were effectively resolved in CDCl3 solution by the addition of the chiral solvating agents (R)-(-)- or (S)-(+)-2,2,2,-trifluoro-1-(9-anthryl)ethanol. Optical purities were determined on the basis of the intensities of the enantiomeric ester methyl proton resonances; the assignment of enantiomeric configurations was based on the relative field positions of these resonances and the examination of molecular models. The analysis of synthetic enantiomeric mixtures of threo-methylphenidate by the proposed NMR method resulted in assay values that agreed closely with the known quantities of each enantiomer in the mixtures tested. The mean +/- SD recovery value for the (2S,2'S)-(-)-threo-enantiomer, amounting to 99.9 +/- 0.6% of added (n = 10), correlated well with that previously found by 1H-NMR spectroscopy with a chiral Eu(III) shift reagent. However, the present approach is simpler, shows less reliance on reagents and solvents of a high purity, and does not require strict anaerobic working conditions.

Stability-indicating proton nuclear magnetic resonance spectroscopic assay method for furosemide in tablets and injections.

A simple, specific, and accurate proton nuclear magnetic resonance (1H-NMR) spectroscopic method has been developed for the identification and assay of furosemide and its degradation product, 4-chloro-5-sulfamoylanthranilic acid (CSA), in tablets and injections. Dissolution of the sample in D2O-NaOD resulted in a solution yielding the required separation among the resonance signals of furosemide, CSA, and tert-butyl alcohol, the internal standard. The mean +/- SD recovery values of furosemide and CSA from 10 synthetic formulations were 99.6 +/- 0.8 and 98.9 +/- 1.7%, respectively. Commercial tablets (6 lots) and injections (5 lots) of furosemide were assayed by the proposed method and found to contain 53.1-99.8% furosemide and 0.3-45.2% CSA.

Optical purity determination of threo-methylphenidate hydrochloride using a chiral europium nuclear magnetic resonance (NMR) shift reagent.

A 1H-NMR spectroscopic method for the determination of the optical purity of threo-methylphenidate hydrochloride is presented. Complexation of the free-base form of the substrate with a chiral Eu(III) shift reagent resulted in two distinct enantiomeric ester methyl proton signals of utility for quantitative work. The accuracy of the method was validated by analyzing six synthetic mixtures of various proportions of (+)- and (-)-threo-methylphenidate. In addition to yielding assay results that were in close agreement with the known weights of each enantiomer in the mixture, and a mean recovery of the (-)-threo-enantiomer of better than 99.0%, the proposed method was capable of accurately measuring as little as 2% of one enantiomer in the presence of the other.

Determination of the enantiomeric composition of carprofen by proton nuclear magnetic resonance spectroscopy with a chiral lanthanide-shift reagent.

The enantiomeric composition of carprofen has been determined in a rapid and reliable manner by proton nuclear magnetic resonance spectroscopy with a chiral lanthanide-shift chelate. Carprofen was converted into a mixture of enantiomeric methyl ester derivatives which were then complexed with tris[3-(heptafluoropropylhydroxymethylene)-(+)-camphorato]europium(III) in CDCl(3). The concentration of substrate in the test sample was 0.15M and the chiral-shift reagent:substrate molar ratio was 0.453. Determination of the enantiomers was based on the relative intensities of the signals for the alpha-methyl protons. The mean recovery +/-SD for six determinations of S(+)-carprofen from synthetic enantiomeric mixtures was 99.3 +/- 1.7%.